Proteomic Biomarkers of Sepsis in Elderly Patients

ABSTRACT

A proteomic expression platform to identify age-related sepsis risk is disclosed using patients with community-acquired pneumonia. A semi-quantitative plasma proteomics workflow was applied which incorporated tandem immuno affinity depletion, iTRAQ labeling, strong cation exchange fractionation, and nanoflow-liquid chromatography coupled to high resolution mass spectrometry. A protein profile was determined that exhibit statistically significant differences in expression levels amongst patients with severe sepsis as a function of age. Representative pathways that are differentially-expressed include, but are not limited to, acute phase response, coagulation signaling, atherosclerosis signaling, lipid metabolism, and production of nitric oxide/reactive oxygen species.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support awarded by the NationalInstitute of General Medical Sciences, National Institutes of Health(grant number R01 GM61992). The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention is related to the field of sepsis. In particular,the invention identifies differences in global protein expression thatare either age-related and/or outcome-related. These differencesrepresent biomarkers to guide risk potential, therapy, monitoring anddiagnosis.

BACKGROUND

Sepsis is a systemic infection that leads to immune dyshomeostasis,organ failure, and in severe cases morbidity. Cohen J, “Theimmunopathgenesis of sepsis” Nature 420:885-891 (2002). Approximately750,000 people develop sepsis annually commonly after an initialdiagnosis of community-acquired pneumonia (CAP). Angus et al.,“Epidemiology of severe sepsis in the United States: analysis ofincidence, outcome, and associated costs of care” Crit Care Med29:1303-1310 (2001). Recently it has been reported that mortality insepsis patients increases with age, although there is no directcorrelation in the expression levels of key cytokines and transcriptionfactors. Martin et al., “The effect of age on the development andoutcome of adult sepsis” Crit Care Med 34:15-21 (2006).

What is needed in the art is a quantitative proteomics platform in orderto identify age- and outcome-related differences in global proteinexpression in plasma obtained from sepsis patients.

SUMMARY

The present invention is related to the field of sepsis. In particular,the invention identifies differences in global protein expression thatare either age-related and/or outcome-related. These differencesrepresent biomarkers to guide risk potential, therapy, monitoring anddiagnosis.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) at least one biological sample derived froman elderly patient suspected of having an infection; ii) at least onepeptide isolated from said at least one biological sample, wherein saidat least one isolated peptide comprises at least one isobaric tag forrelative and absolute quantitation (iTRAQ) reporter ion; iii) a systemcomprising a liquid chromatography column and a mass spectrometer; andiv) a control proteomic pathway expression profile; b) contacting saidat least one iTRAQ-peptide with said system to create at least oneiTRAQ-peptide analysis spectrum; c) processing said at least oneiTRAQ-peptide analysis spectrum to create a sepsis proteomic pathwayexpression profile, wherein said sepsis proteomic pathway expressionprofile comprises at least one over-expressed protein pathway ascompared to said control proteomic pathway expression profile; and d)diagnosing said patient with severe sepsis upon identification of saidover-expressed protein pathway. In one embodiment, the over-expressedprotein pathway is a liver retinoid X receptor activation pathway. Inone embodiment, the liver retinoid X receptor activation pathway isabout ten-fold over-expressed as compared to said control proteomicpathway expression profile. In one embodiment, the over-expressedprotein pathway is an acute phase response signaling pathway. In oneembodiment, the acute phase response signaling pathway is abouteight-fold over-expressed as compared to said control proteomic pathwayexpression profile. In one embodiment, the over-expressed proteinpathway is an atherosclerosis signaling pathway. In one embodiment, theatherosclerosis signaling pathway is about seven-fold over-expressed ascompared to said control proteomic pathway expression profile. In oneembodiment, the over-expressed protein pathway is an interleukin-2signaling pathway. In one embodiment, the interleukin-2 signalingpathway is about seven-fold over-expressed as compared to said controlproteomic pathway expression profile. In one embodiment, theover-expressed protein pathway is a nitric oxide/oxygen reactive speciespathway. In one embodiment, the nitric oxide/oxygen reactive speciespathway is about seven-fold over-expressed as compared to said controlproteomic pathway expression profile. In one embodiment, theover-expressed protein pathway is a clathrin-mediated endocytosissignaling pathway. In one embodiment, the clathrin-mediated endocytosissignaling pathway is about seven-fold over-expressed as compared to saidcontrol proteomic pathway expression profile. In one embodiment, theover-expressed protein pathway is an lipopolysacharride/interleukin-1retinoid X receptor inhibition pathway. In one embodiment, thelipopolysacharride/interleukin-1 retinoid X receptor inhibition pathwayis about three-fold over-expressed as compared to said control proteomicpathway expression profile. In one embodiment, the over-expressedprotein pathway is an farnesoid X receptor activation pathway. In oneembodiment, the farnesoid X receptor activation pathway is abouttwo-fold over-expressed as compared to said control proteomic pathwayexpression profile. In one embodiment, the over-expressed proteinpathway is a hepatic stellate cell activation pathway. In oneembodiment, the hepatic stellate cell activation pathway is abouttwo-fold over-expressed as compared to said control proteomic pathwayexpression profile. In one embodiment, the over-expressed proteinpathway is an actin cytoskeleton signaling pathway. In one embodiment,the actin cytoskeleton signaling pathway is about two-foldover-expressed as compared to said control proteomic pathway expressionprofile.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) at least one biological sample derived froma young patient suspected of having an infection; ii) at least onepeptide isolated from said at least one biological sample, wherein saidat least one isolated peptide comprises at least one isobaric tag forrelative and absolute quantitation (iTRAQ) reporter ion; iii) a systemcomprising a liquid chromatography column and a mass spectrometer; andiv) a control proteomic pathway expression profile; b) contacting saidat least one iTRAQ-peptide with said system to create at least oneiTRAQ-peptide analysis spectrum; c) processing said at least oneiTRAQ-peptide analysis spectrum to create a sepsis proteomic pathwayexpression profile, wherein said sepsis proteomic pathway expressionprofile comprises at least one over-expressed protein pathway ascompared to said control proteomic pathway expression profile; and d)diagnosing said patient with severe sepsis upon identification of saidover-expressed protein pathway. In one embodiment, the over-expressedprotein pathway is a liver retinoid X receptor activation pathway. Inone embodiment, the liver retinoid X receptor pathway is aboutsixteen-fold over-expressed as compared to said control proteomicpathway expression profile. In one embodiment, the over-expressedprotein pathway is an acute phase response signaling pathway. In oneembodiment, the acute phase response signaling pathway is aboutsixteen-fold over-expressed as compared to said control proteomicpathway expression profile. In one embodiment, the over-expressedprotein pathway is an atherosclerosis signaling pathway. In oneembodiment, the atherosclerosis signaling pathway is about ten-foldover-expressed as compared to said control proteomic pathway expressionprofile. In one embodiment, the over-expressed protein pathway is aninterleukin-2 signaling pathway. In one embodiment, the interleukin-2signaling pathway is about ten-fold over-expressed as compared to saidcontrol proteomic pathway expression profile. In one embodiment, theover-expressed protein pathway is a nitric oxide/oxygen reactive speciespathway. In one embodiment, the nitric oxide/oxygen reactive speciespathway is about ten-fold over-expressed as compared to said controlproteomic pathway expression profile. In one embodiment, theover-expressed protein pathway is a clathrin-mediated endocytosissignaling pathway. In one embodiment, the clathrin-mediated endocytosissignaling pathway is about eleven-fold over-expressed as compared tosaid control proteomic pathway expression profile. In one embodiment,the over-expressed protein pathway is a blood factor coagulationpathway. In one embodiment, the blood factor coagulation is aboutseven-fold over-expressed as compared to said control proteomic pathwayexpression profile. In one embodiment, the over-expressed proteinpathway is an extrinsic prothrombin activation pathway. In oneembodiment, the extrinsic prothrombin activation pathway is aboutfive-fold over-expressed as compared to said control proteomic pathwayexpression profile. In one embodiment, the over-expressed proteinpathway is an intrinsic prothrombin activation pathway. In oneembodiment, the intrinsic prothrombin activation pathway is aboutfive-fold over-expressed as compared to said control proteomic pathwayexpression profile. In one embodiment, the over-expressed proteinpathway is a farnesoid X receptor activation pathway. In one embodiment,the farnesoid X receptor activation pathway is about five-foldover-expressed as compared to said control proteomic pathway expressionprofile. In one embodiment, the over-expressed protein pathway is anlipopolysaccharide/interleukin-1 retinoid X receptor inhibition pathway.In one embodiment, the lipopolysaccharide/interleukin-1 retinoid Xreceptor inhibition pathway is about six-fold over-expressed as comparedto said control proteomic pathway expression profile.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) at least one biological sample derived fromat least one patient suspected of having an infection; ii) at least onepeptide isolated from said at least one biological sample, wherein saidat least one isolated peptide comprises at least one isobaric tag forrelative and absolute quantitation (iTRAQ) reporter ion; and iii) asystem comprising a liquid chromatography column and a massspectrometer; b) contacting said at least one iTRAQ-peptide with saidsystem to create at least one iTRAQ-peptide analysis spectrum; c)processing said at least one iTRAQ-peptide analysis spectrum to create afirst proteomic pathway expression profile and a second proteomicexpression profile, wherein said first proteomic pathway expressionprofile comprises at least one differentially expressed protein pathwayas compared to said second proteomic pathway expression profile; and d)diagnosing said patient with severe sepsis upon identification of saiddifferentially expressed protein pathway. In one embodiment, thedifferentially expressed proteomic pathway comprises an acute phaseresponse pathway. In one embodiment, the acute phase response pathway isdifferentially expressed by a ratio ranging between 8:1-16:1. In oneembodiment, the differentially expressed proteomic pathway comprises ablood factor coagulation pathway. In one embodiment, the blood factorcoagulation pathway is differentially expressed by a ratio rangingbetween 4:1-5:1. In one embodiment, the differentially expressedproteomic pathway comprises a lipid metabolism pathway. In oneembodiment, the lipid metabolism pathway is differentially expressed bya ratio ranging between 6:1-10:1. In one embodiment, the differentiallyexpressed proteomic pathway comprises an interleukin pathway. In oneembodiment, the interleukin pathway is differentially over-expressed bya ratio ranging between 2:1-10:1. In one embodiment, the differentiallyexpressed proteomic pathway comprises a nitric oxide/reactive oxygenspecies pathway. In one embodiment, the nitric oxide/reactive oxygenspecies pathway is differentially over-expressed by a ratio rangingbetween 7:1-10:1. In one embodiment, the at least one patient is anelderly patient without severe sepsis. In one embodiment, the at leastone patient is a young patient without severe sepsis. In one embodiment,the at least one patient is an elderly patient with severe sepsis. Inone embodiment, the at least one patient is a young patient with severesepsis. In one embodiment, the infection comprises community acquiredpneumonia. In one embodiment, the method further comprises treating eachof said at least one patient differentially based upon said at least onesepsis proteomic expression profile.

In one embodiment, the present invention contemplates a method forage-related sepsis treatment, comprising: a) providing; i) an olderpatient exhibiting at least one sepsis symptom and differentiallyexpressing at least one protein pathway as compared to a youngerpatient; and ii) a pharmaceutical compound that interacts with aspecific drug target of said at least one differentially expressedprotein pathway; b) administering said pharmaceutical compound to saidpatient under conditions such that said at least one sepsis symptom isreduced. In one embodiment, the at least one differentially expressedprotein pathway comprises a hepatic stellate cell activation pathway. Inone embodiment, the at least one differentially expressed proteinpathway comprises an actin cytoskeleton signaling pathway. In oneembodiment, the method further comprises a combination treatment of saidpharmaceutical compound with a conventional sepsis treatment. In oneembodiment, the conventional sepsis treatment is selected from the groupconsisting of antibiotics, inflammatory mediator inhibitors, steroids,proinflammatory cytokine inhibitors, statins, coagulation cascadeinhibitors and immunostimulators. In one embodiment, the older patientranges in age between 70-85 years old. In one embodiment, the youngerpatient ranges in age between 50-65 years old.

In one embodiment, the present invention contemplates a method forage-related sepsis treatment, comprising: a) providing; i) a youngerpatient exhibiting at least one sepsis symptom and differentiallyexpressing at least one protein pathway as compared to an older patient;and ii) a pharmaceutical compound that interacts with a specific drugtarget of said at least one differentially expressed protein pathway; b)administering said pharmaceutical compound to said patient underconditions such that said at least one sepsis symptom is reduced. In oneembodiment, the at least one differentially expressed protein pathwaycomprises a blood factor coagulation pathway. In one embodiment, the atleast one differentially expressed protein pathway comprises anextrinsic prothrombin activation pathway. In one embodiment, the atleast one differentially expressed protein pathway comprises anintrinsic prothrombin activation pathway. In one embodiment, the methodfurther comprises a combination treatment of said pharmaceuticalcompound with a conventional sepsis treatment. In one embodiment, theconventional sepsis treatment is selected from the group consisting ofantibiotics, inflammatory mediator inhibitors, steroids, proinflammatorycytokine inhibitors, statins, coagulation cascade inhibitors andimmunostimulators. In one embodiment, the younger patient ranges in agebetween 50-65 years old. In one embodiment, the older patient ranges inage between 70-85 years old.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) at least one peptide isolated from at leastone sepsis biological sample, wherein said at least one isolated peptidecomprises at least one isobaric tag for relative and absolutequantitation (iTRAQ) reporter ion; and iii) a system comprising a liquidchromatography column and a mass spectrometer; b) contacting said atleast one iTRAQ-peptide with said system to create at least oneiTRAQ-peptide analysis spectrum; and c) processing said at least oneiTRAQ-peptide analysis spectrum to create a first proteomic pathwayexpression profile and a second proteomic expression profile, whereinsaid first proteomic pathway expression profile comprises at least onedifferentially expressed protein pathway as compared to said secondproteomic pathway expression profile. In one embodiment, thedifferentially expressed proteomic pathway comprises an acute phaseresponse pathway. In one embodiment, the acute phase response pathway isdifferentially expressed by a ratio ranging between 8:1-16:1. In oneembodiment, the differentially expressed proteomic pathway comprises ablood factor coagulation pathway. In one embodiment, the blood factorcoagulation pathway is differentially expressed by a ratio rangingbetween 4:1-5:1. In one embodiment, the differentially expressedproteomic pathway comprises a lipid metabolism pathway. In oneembodiment, the lipid metabolism pathway is differentially expressed bya ratio ranging between 6:1-10:1. In one embodiment, the differentiallyexpressed proteomic pathway comprises an interleukin pathway. In oneembodiment, the interleukin pathway is differentially over-expressed bya ratio ranging between 2:1-10:1. In one embodiment, the differentiallyexpressed proteomic pathway comprises a nitric oxide/reactive oxygenspecies pathway. In one embodiment, the nitric oxide/reactive oxygenspecies pathway is differentially over-expressed by a ratio rangingbetween 7:1-10:1.

DEFINITIONS

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity but also plural entities and also includes thegeneral class of which a specific example may be used for illustration.The terminology herein is used to describe specific embodiments of theinvention, but their usage does not delimit the invention, except asoutlined in the claims.

The term “about” as used herein, in the context of any of any assaymeasurements refers to +/−5% of a given measurement.

The term “substitute for” as used herein, refers to the switching theadministration of a first compound or drug to a subject for a secondcompound or drug to the subject.

The term “suspected of having”, as used herein, refers a medicalcondition or set of medical conditions (e.g., preliminary symptoms)exhibited by a patient that is insufficient to provide a differentialdiagnosis. Nonetheless, the exhibited condition(s) would justify furthertesting (e.g., autoantibody testing) to obtain further information onwhich to base a diagnosis.

The term “isobaric tag for relative and absolute quantitation (iTRAQ)”refers to a non-gel-based technique used to quantify proteins fromdifferent sources in a single experiment. It uses isotope-coded covalenttags. iTRAQ is used in proteomics to study quantitative changes in theproteome. Ross et al., “Multiplexed protein quantitation in saccaromycescerevisiae using amine-reactive isobaric tagging reagents” Molecular &Cellular Proteomics 3:1154-1169 (2004). iTRAQ is based on the covalentlabeling of the N-terminus and side chain amines of peptides fromprotein digestions with tags of varying mass. For example, two suchreagents include, but are not limited to a 4-plex and an 8-plex, whichcan be used to label all peptides from different samples/treatments.These samples are then pooled and usually fractionated by nano liquidchromatography and analyzed by tandem mass spectrometry (MS/MS). Adatabase search is then performed using the fragmentation data toidentify the labeled peptides and hence the corresponding proteins. Thefragmentation of the attached tag generates a low molecular massreporter ion that can be used to relatively quantify the peptides andthe proteins from which they originated (e.g., an iTRAQ-peptide analysisspectrum).

The term “infection” as used herein, refers to an invasion of a hostorganism's body tissues by disease-causing organisms, theirmultiplication, and the reaction of host tissues to these organisms andthe toxins they produce. Infectious diseases, also known astransmissible diseases or communicable diseases, comprise clinicallyevident illness (i.e., characteristic medical signs and/or symptoms ofdisease) resulting from the infection, presence and growth of pathogenicbiological agents in an individual host organism. For example, infectionby a virus may result in acquired community pneumonia that can developinto sepsis.

The term “community-acquired pneumonia (CAP)” as used herein, refers toa type of pneumonia (any of several lung diseases) acquired infectiouslyfrom normal social contact (that is, in the community) as opposed tobeing acquired during hospitalization (hospital-acquired pneumonia). Incommunity-acquired pneumonia, individuals who have not recently beenhospitalized develop an infection of the lungs (pneumonia). CAP is acommon illness and can affect people of all ages. CAP often causesproblems such as difficulty breathing, fever, chest pains, and a coughand sometime develops into sepsis. CAP occurs because the areas of thelung that absorb oxygen (alveoli) from the atmosphere become filled withfluid and cannot work effectively.

The term “liquid chromatography” as used herein, refers a set oflaboratory techniques for the separation of mixtures generally using acolumn (e.g., a liquid chromatography column). A mixture of compoundsmay be dissolved in a “liquid”, which carries the mixture through acolumn comprising a stationary phase matrix. The various compounds ofthe mixture travel at different speeds, causing them to separate.

The term “mass spectrometer” as used herein, refers to a massspectrometer comprising three components such as an ion source, a massanalyzer, and a detector. The ionizer converts a portion of the sampleinto ions. There is a wide variety of ionization techniques, dependingon the phase (solid, liquid, gas) of the sample and the efficiency ofvarious ionization mechanisms for the unknown species. An extractionsystem removes ions from the sample, which are then trajected throughthe mass analyzer and onto the detector. The differences in masses ofthe fragments allows the mass analyzer to sort the ions by theirmass-to-charge ratio. The detector measures the value of an indicatorquantity and thus provides data for calculating the abundances of eachion present. Some detectors also give spatial information, e.g., amultichannel plate.

The term “tandem mass spectrometer” as used herein, refers to a type ofmass spectrometer that is capable of multiple rounds of massspectrometry, usually separated by some form of molecule fragmentation.For example, one mass analyzer can isolate one peptide from manyentering a mass spectrometer. A second mass analyzer then stabilizes thepeptide ions while they collide with a gas, causing them to fragment bycollision-induced dissociation (CID). A third mass analyzer then sortsthe fragments produced from the peptides. Tandem MS can also be done ina single mass analyzer over time, as in a quadrapole ion trap. There arevarious methods for fragmenting molecules for tandem MS, includingcollision-induced dissociation (CID), electron capture dissociation(ECD), electron transfer dissociation (ETD), infrared multiphotondissociation (IRMPD), blackbody infrared radiative dissociation (BIRD),electron-detachment dissociation (EDD) and surface-induced dissociation(SID). An important application using tandem mass spectrometry is inprotein identification.

The term “proteomics” as used herein, refers to a large-scale study ofproteins, particularly their structures and functions. A proteome maycomprise an entire set of proteins produced or modified by an organismor system within that organism. Such organismic systems may comprisespecific protein pathways whose participating proteins/peptides areco-regulated. While proteomics generally refers to the large-scaleexperimental analysis of proteins, it is often specifically used insupport of protein purification and mass spectrometry analysis.

The term “proteomic pathway expression profile” as used herein, refersto an analysis of protein expression on a large scale. Proteomicexpression analysis helps identify proteins that are differentiallyexpressed in different biological samples-such as diseased vs. healthytissue, or tissue from a young patient vs. an elderly patient. If aprotein is found only in a tissue sample from an elderly patient then itcan be a useful drug target or diagnostic marker. Proteins with same orsimilar expression profiles may also be functionally related. There aretechnologies such as 2D-PAGE and mass spectrometry that are used inexpression proteomics. Two or more different proteomic pathwayexpression profiles may be compared for differential protein pathwayexpression. For example, a simple ratio calculation of the processeddata from an iTRAQ analysis can determine whether one protein isover/under-expressed. In most cases herein, a ratio is determinedbetween a sepsis proteomic pathway expression profile (e.g., where thebiological sample is derived from a subject suspected of having aninfection) and a control proteomic pathway expression profile (e.g.,where the biological sample is derived from a subject that is notsuspected of having a medical condition).

The term “symptom”, as used herein, refers to any subjective orobjective evidence of disease or physical disturbance observed by thepatient. For example, subjective evidence is usually based upon patientself-reporting and may include, but is not limited to, pain, headache,visual disturbances, nausea and/or vomiting. Alternatively, objectiveevidence is usually a result of medical testing including, but notlimited to, body temperature, complete blood count, lipid panels,thyroid panels, blood pressure, heart rate, electrocardiogram, tissueand/or body imaging scans.

The term “disease” or “medical condition”, as used herein, refers to anyimpairment of the normal state of the living animal or plant body or oneof its parts that interrupts or modifies the performance of the vitalfunctions. Typically manifested by distinguishing signs and symptoms, itis usually a response to: i) environmental factors (as malnutrition,industrial hazards, or climate); ii) specific infective agents (asworms, bacteria, or viruses); iii) inherent defects of the organism (asgenetic anomalies); and/or iv) combinations of these factors.

The term “drug” or “compound” as used herein, refers to anypharmacologically active substance capable of being administered whichachieves a desired effect. Drugs or compounds can be synthetic ornaturally occurring, non-peptide, proteins or peptides, oligonucleotidesor nucleotides, polysaccharides or sugars.

The term “administered” or “administering”, as used herein, refers toany method of providing a composition to a patient such that thecomposition has its intended effect on the patient. An exemplary methodof administering is by a direct mechanism such as, local tissueadministration (i.e., for example, extravascular placement), oralingestion, transdermal patch, topical, inhalation, suppository etc.

The term “effective amount” as used herein, refers to a particularamount of a pharmaceutical composition comprising a therapeutic agentthat achieves a clinically beneficial result (i.e., for example, areduction of symptoms). Toxicity and therapeutic efficacy of suchcompositions can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio LD50/ED50. Compounds that exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and additional animal studies can be used in formulatinga range of dosage for human use. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,”“prevent” and grammatical equivalents (including “lower,” “smaller,”etc.) when in reference to the expression of any symptom in an untreatedsubject relative to a treated subject, mean that the quantity and/ormagnitude of the symptoms in the treated subject is lower than in theuntreated subject by any amount that is recognized as clinicallyrelevant by any medically trained personnel. In one embodiment, thequantity and/or magnitude of the symptoms in the treated subject is atleast 10% lower than, at least 25% lower than, at least 50% lower than,at least 75% lower than, and/or at least 90% lower than the quantityand/or magnitude of the symptoms in the untreated subject.

The term “inhibitory compound” as used herein, refers to any compoundcapable of interacting with (i.e., for example, attaching, binding etc)to a binding partner under conditions such that the binding partnerbecomes unresponsive to its natural ligands. Inhibitory compounds mayinclude, but are not limited to, small organic molecules, antibodies,and proteins/peptides.

The term “patient” or “subject”, as used herein, is a human or animaland need not be hospitalized. For example, out-patients, persons innursing homes are “patients.” A patient may comprise any age of a humanor non-human animal and therefore includes both adult and juveniles(i.e., children). It is not intended that the term “patient” connote aneed for medical treatment, therefore, a patient may voluntarily orinvoluntarily be part of experimentation whether clinical or in supportof basic science studies.

The term “affinity” as used herein, refers to any attractive forcebetween substances or particles that causes them to enter into andremain in chemical combination. For example, an inhibitor compound thathas a high affinity for a receptor will provide greater efficacy inpreventing the receptor from interacting with its natural ligands, thanan inhibitor with a low affinity.

The term “derived from” as used herein, refers to the source of acompound or sequence. In one respect, a compound or sequence may bederived from an organism or particular species. In another respect, acompound or sequence may be derived from a larger complex or sequence.

The term “protein” as used herein, refers to any of numerous naturallyoccurring extremely complex substances (as an enzyme or antibody) thatconsist of amino acid residues joined by peptide bonds, contain theelements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general,a protein comprises amino acids having an order of magnitude within thehundreds.

The term “peptide” as used herein, refers to any of various amides thatare derived from two or more amino acids by combination of the aminogroup of one acid with the carboxyl group of another and are usuallyobtained by partial hydrolysis of proteins. In general, a peptidecomprises amino acids having an order of magnitude with the tens.

The term “polypeptide”, refers to any of various amides that are derivedfrom two or more amino acids by combination of the amino group of oneacid with the carboxyl group of another and are usually obtained bypartial hydrolysis of proteins. In general, a peptide comprises aminoacids having an order of magnitude with the tens or larger.

The term “pharmaceutically” or “pharmacologically acceptable”, as usedherein, refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human.

The term, “pharmaceutically acceptable carrier”, as used herein,includes any and all solvents, or a dispersion medium including, but notlimited to, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils, coatings, isotonic and absorption delayingagents, liposome, commercially available cleansers, and the like.Supplementary bioactive ingredients also can be incorporated into suchcarriers.

The term, “purified” or “isolated”, as used herein, may refer to apeptide composition that has been subjected to treatment (i.e., forexample, fractionation) to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the protein or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 90%, about 95% or more of the composition(i.e., for example, weight/weight and/or weight/volume). The term“purified to homogeneity” is used to include compositions that have beenpurified to ‘apparent homogeneity” such that there is single proteinspecies (i.e., for example, based upon SDS-PAGE or HPLC analysis). Apurified composition is not intended to mean that some trace impuritiesmay remain.

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and more preferably 90% free from other componentswith which they are naturally associated. An “isolated polynucleotide”is therefore a substantially purified polynucleotide.

The term “sample” as used herein is used in its broadest sense andincludes environmental and biological samples. Environmental samplesinclude material from the environment such as soil and water. Biologicalsamples may be animal, including, human, fluid (e.g., blood, plasma andserum), solid (e.g., stool), tissue, liquid foods (e.g., milk), andsolid foods (e.g., vegetables). For example, a pulmonary sample may becollected by bronchoalveolar lavage (BAL) which comprises fluid andcells derived from lung tissues. A biological sample may comprise acell, tissue extract, body fluid, chromosomes or extrachromosomalelements isolated from a cell, genomic DNA (in solution or bound to asolid support such as for Southern blot analysis), RNA (in solution orbound to a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like.

The term “bind” as used herein, includes any physical attachment orclose association, which may be permanent or temporary. Generally, aninteraction of hydrogen bonding, hydrophobic forces, van der Waalsforces, covalent and ionic bonding etc., facilitates physical attachmentbetween the molecule of interest and the analyte being measuring. The“binding” interaction may be brief as in the situation where bindingcauses a chemical reaction to occur. That is typical when the bindingcomponent is an enzyme and the analyte is a substrate for the enzyme.Reactions resulting from contact between the binding agent and theanalyte are also within the definition of binding for the purposes ofthe present invention.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 illustrates one embodiment for a parallel blood plasma workflowschematic using four iTRAQ reporter ions in Multiple Affinity RemovalSystem (MARS) columns in preparation for nanoscale liquid chromatographycoupled to tandem mass spectrometry (Nano-LC-MS/MS) analysis. ExemplaryiTRAQ Reporter Ions-Column A: iTRAQ_114 (I₁₁₄); Column B: iTRAQ_115(I₁₁₅); Column C: iTRAQ_116 (I₁₁₆); Column D: iTRAQ_117 (I₁₁₇).

FIG. 2 shows exemplary data over the course of ten (10) Nano-LC-MS/MSexperiments.

FIG. 2A: Solid Line: Accumulation of identified proteins (773) of which726 proteins were identified. Bars:

FIG. 2B: Data demonstrating the relative stability of spectral countsover the course of the ten Nano-LC-MS/MS experiments.

FIG. 3 presents Venn diagrams depicting the differences and overlapbetween the various subject populations.

FIG. 4 presents a present understanding of a physiological process ofthrombosis initiated by sepsis.

FIG. 5 presents a present understanding of a cytokine regulationpathway.

FIG. 6 presents exemplary data showing differentially expressed pathwaysbetween the YY/OY subject populations.

FIG. 7 presents exemplary data showing differentially expressed pathwaysbetween the YN/ON subject populations.

FIG. 8 presents exemplary data showing differentially expressed pathwaysbetween the YY/YN subject populations.

FIG. 9 presents exemplary data showing differentially expressed pathwaysbetween the OY/ON subject populations.

FIG. 10 presents exemplary data, in triplicate, showing thereproducibility of Nano-LC-MS/MS.

FIG. 10A: A MARS-depleted human plasma chromatogram.

FIG. 10B: A tandem MARS-depleted human plasma chromatogram.

FIG. 11 presents exemplary data showing the reproducibility of theworkflow using four plasma samples from CAP patients that were preparedand analyzed with the iTRAQ workflow. Across the triplicate sample sets,90% of the quantified proteins had a RSD smaller than 0.25. BlueCross-Hatched Bar: I₁₁₅/I₁₁₄ protein number, Orange Cross-Hatched Bar:I₁₁₆/I₁₁₄ protein number, and Green Cross-Hatched Bar: I₁₁₇/I₁₁₄ proteinnumber. Blue Squares: I₁₁₅/I₁₁₄ cumulative frequency. Orange Squares:I₁₁₆/I₁₁₄ cumulative frequency. Green Squares: I₁₁₇/I₁₁₄ cumulativefrequency.

FIG. 12 presents exemplary data showing a distribution of protein ratiosacross four plasma samples as described in FIG. 11. Blue Bars:I₁₁₅/I₁₁₄; Green Bars: I₁₁₆/I₁₁₄; Red Bars: I₁₁₇/I₁₁₄.

FIG. 13 presents the identification of over-expressed protein pathwaysbased upon the quantified protein ratios.

FIG. 14 presents exemplary MS/MS spectrum data of the fragmentationpattern of the peptide D(iTRAQ)LATVYVDVLK(iTRAQ) (z=+2, m/z+763.4244)from apoliprotein A-1 showing how protein ratios were obtained based onthe corresponding peptide ratios as the same peptides from fourdifferent samples cannot be differentiated on MS level. Consequently,peptide identification and peptide quantitation were based on MS/MSspectrum obtained from CID and HCD fragmentation, respectively. Proteinidentification: CID MS/MS. Protein Quantitation: HCD MS/MS.

FIG. 14A: Conventional MS spectra.

FIG. 14B: CID MS/MS spectra and analysis.

FIG. 14C; HCD MS/MS spectra and analysis.

FIG. 14D: Control spectra showing the reporter ion ratios(I₁₁₄:I₁₁₅:I₁₁₆:I₁₁₇=1:3.44:2.62:1.45).

FIG. 15A presents conventional understanding of a sepsis pathway. CohenJ., Nature420:885-891 (2002).

FIG. 15B presents a representative distribution of the relationshipbetween age and severe sepsis. Kale et al., PLoSone 5:e13852 (2010).

FIG. 16 presents representative data showing sepsis-induced cytokinepatterns as a function of age. Blue Diamonds: <50 years. Red Squares:50-64 years. Green Triangles: 65-74 years. Purple Crosses: 75-84 years.Blue Crosses: >85 years.

FIG. 16A: Tissue necrosis factor (TNF)

FIG. 16B: Interleukin 6 (IL-6)

FIG. 16C: Interleukin 10 (IL-10).

FIG. 17 presents representative Nano-LC-MS/MS data for a peptidefragment (A(iTRAQ)EFQDALEK(iTRAQ)+2H)²⁺ of Complement C4. SpectralCounts=450; Unique peptides=59.

FIG. 17A: A CID spectra and analysis.

FIG. 17B: A HCD spectra and analysis.

FIG. 18 presents representative Nano-LC-MS/MS data for a peptidefragment (A(iTRAQ)EFQDALEK(iTRAQ)+2H)²⁺ of Complement C4. SpectralCount=450; Unique peptides=59, Ratio 117/114=1.14±0.38.

FIG. 18A: A CID spectra and analysis.

FIG. 18B: A HCD spectra and analysis.

FIG. 19 presents exemplary data showing the reliability of Nano-LC-MS/MSquantitation (HCD) by peptide-to-protein inferencing of Complement C4data in FIGS. 17 and 18.

FIG. 19A: Peptide ratio distribution.

FIG. 19B: Protein ratio distribution.

FIG. 20 presents representative Nano-LC-MS/MS data for a peptidefragment (D(iTRAQ)LATVYVDVLK(iTRAQ)+2H)²⁺ of apolipoprotein A-1.Spectral Counts=1734; Unique peptides=24.

FIG. 20A: A CID spectra and analysis.

FIG. 20B: A HCD spectra and analysis.

FIG. 21 presents representative Nano-LC-MS/MS data for a peptidefragment (D(iTRAQ)LATVYVDVLK(iTRAQ)+2H)²⁺ of apolipoprotein A-1.Spectral Counts=1734; Unique peptides=24.

FIG. 21A: A CID spectra and analysis.

FIG. 21B: A HCD spectra and analysis.

FIG. 22 presents an exemplary LC chromatogram subsequent to SCXfractionation.

FIG. 22A: LC chromatograms for individual SCX fractions analyzed intriplicate.

FIG. 22B: Exemplary mass spectra of peptides eluted from SCX fraction 7(t_(r)=57.20 min with m/z 748.8046);

FIG. 22C: Exemplary mass spectra of peptides eluted from SCX fraction 6(t_(r)=37.26 min with m/z 742.9010).

FIG. 22D: Exemplary CID MS/MS of peptides eluted from SCX fraction 7.

FIG. 22E: Exemplary CID MS/MS of peptides eluted from SCX fraction 6.

FIG. 22F: Exemplary HCD MS/MS of peptides eluted from SCX fraction 7(inset: low m/z region).

FIG. 22G: Exemplary HCD MS/MS of peptides eluted from SCX fraction 6(inset: low m/z region).

FIG. 23 presents exemplary data showing an average number±standarddeviation of proteins (light bars) and spectral counts (dark bars)identified in each experiment. The cumulative number of proteins(diamonds) identified with each subsequent experiment is also shown.

FIG. 24 presents exemplary data showing Western blotting images andhistogram displaying a normalized intensity±standard deviation (n=6) ofthe proteins across each group. The intensity for each individual bandis normalized to the total intensity of the blot.

FIG. 24A: C-reactive protein,

FIG. 24B: Fibrinogen alpha chain.

FIG. 24C: Apolipoprotein CIII.

FIG. 25 presents an illustrative Venn diagram ofdifferentially-expressed proteins for each age group (i.e., YS/YC 50-65years old and OS/OC 70-85 years old). The number of proteins that havehigher (↑) or lower (↓) fold-change values in each comparison are alsoshown.

FIG. 26 presents exemplary data showing a histogram plot of biologicalpathways associated with differentially expressed proteins as a functionof severity of sepsis (N=59). The p value cutoff for the IngenuityPathway Analysis (IPA) is p<0.05.

DETAILED DESCRIPTION

The present invention is related to the field of sepsis. In particular,the invention identifies differences in global protein expression thatare either age-related and/or outcome-related. These differencesrepresent biomarkers to guide risk potential, therapy, monitoring anddiagnosis.

In one embodiment, the present invention contemplates a method forproducing a sepsis proteomic expression profile comprising MARS-sampledepletion, iTRAQ reporter ions, SCX protein fractionation andnano-LC-MS/MS. In one embodiment, the method generates highlyreproducible data with 90% of RSD smaller than 0.25.

I. Sepsis

Sepsis is believed to be a systematic inflammatory state triggered byinfection. Other conditions including, but not limited to, multipleorgan failure and hypoperfusion (e.g., hypotension, decreased urineoutput) in combination with sepsis can result in severe sepsis.Dellinger et al., “Surviving Sepsis Campaign: International Guidelinesfor Management of Severe Sepsis and Septic Shock: 2012” Critical CareMedicine 41:580-637 (2013). One of the leading causes of sepsis iscommunity-acquired pneumonia (CAP). Kale et al., “The effects of age oninflammatory and coagulation-fibrinolysis response in patientshospitalized for pneumonia” PLoS ONE 5:e13852 (2010); Dremsizov et al.,“Severe sepsis in community-acquired pneumonia” Chest 129:968-978(2006); and Beutz et al., “Community-acquired pneumonia and sepsis”Clinics In Chest Medicine 26:19-28 (2005). The presence and severity oforgan failure is one of the most important determinants of mortalityfollowing sepsis. Severe sepsis affects ˜750,000 persons annually in theUnited States and is one of the most common causes of death in intensivecare units. Vincent et al., “SOFA so good for predicting long-termoutcomes” Resuscitation 83:537-538 ((2012)); Carrigan et al., “Towardresolving the challenges of sepsis diagnosis” Clinical Chemistry50:1301-1314 ((2004); Davis, B. H., “Improved diagnostic approaches toinfection/sepsis detection” Expert Review of Molecular Diagnostics5:193-207 (2005); Levy et al., “2001 SCCM/ESICM/ACCP/ATS/SISInternational Sepsis Definitions Conference” Critical Care Medicine31:1250-1256 (2003); Balk, R. A., “Severe sepsis and septic shock:definitions, epidemiology, and clinical manifestations” Critical CareClinics 16:179-192 (2000); Martin et al., “The epidemiology of sepsis inthe United States from 1979 through 2000” New England Journal ofMedicine 348:1546-1554 (2003); Cohen, J., “The immunopathogenesis ofsepsis” Nature 420:885-891 (2002).

It has been reported that the incidence of severe sepsis and mortalityrate increases sharply after the age of 65. Angus et al., “Epidemiologyof severe sepsis in the United States: Analysis of incidence, outcome,and associated costs of care” Critical Care Medicine 29:1303-1310(2001); and Martin et al., “The effect of age on the development andoutcome of adult sepsis” Critical Care Medicine 34:15-21 (2006. Forexample, CAP patients ≧85 years old have a 2.8-fold increased risk ofsevere sepsis and a 17-fold increased mortality rate compared topatients ≦50 years old. The notable age-related differences in severesepsis risk may be explained in part by immunosenescence underlyingchronic disease. Immunosenesence is manifested in the elderly throughdecreased numbers of T cells, impaired B-cell function, increasedapoptosis of neutrophils, and reduced bactericidal response ofmacrophages. Opal et al., “The immunopathogenesis of sepsis in elderlypatients” Clinical Infectious Diseases 41:S504-S512 (2005); and Wick etal., “The aging immune system: primary and secondary alterations ofimmune reactivity in the elderly” Experimental Gerontology 32:401-413(1997).

Most of the above studies that examined age-related differences inimmune response using animal models and humans have largely focused oneither the adaptive immune response or select pathways (inflammatory,coagulation, and fibrinolysis markers) in the innate immune response. Acomprehensive assessment of differences in the immune response has notbeen conducted. Trials testing immunomodulating therapies for sepsis inbroad populations have failed to consistently improve outcomes of sepsispatients. Hotchkiss et al., “The pathophysiology and treatment ofsepsis” New England Journal of Medicine 348:1.38-150 (2003). Analternative approach is to personalize sepsis therapies based on hostcharacteristics, such as age. To further the development of such apersonalized approach, a better understanding of the differences inimmune response due to age is necessary.

In sepsis, blood pressure generally drops resulting in shock. Majororgans and body systems, including the kidneys, liver, lungs, andcentral nervous system, stop working properly because of poor bloodflow. A change in mental status and very fast breathing may be theearliest signs of sepsis. In general, symptoms of sepsis can include,but are not limited to, chills, confusion or delirium, fever or low bodytemperature (hypothermia), light-headedness due to low blood pressure,rapid heartbeat, shaking, skin rash, warm skin, bruising and/orbleeding.

II. Nanoscale Liquid Chromatography Coupled to Tandem Mass Spectrometry

Nanoscale liquid chromatography coupled to tandem mass spectrometry(nano LC-MS/MS) has been widely implemented in the field of proteomics.In fact, its sensitivity has advantages over conventional LC-MS/MS thatallow the analysis of peptide mixtures in sample-limited situations(e.g., proteolytically digested proteins isolated by two-dimensional gelelectrophoresis). Technical challenges, associated with low flow ratesof the chromatographic separation, make this technology still difficultto run routinely. Some a nano LC-MS/MS setups allow several weeks ofcontinuous operation for the analysis of peptides derived by enzymaticdigestion of either purified proteins or moderately complex proteinmixtures. Gaspari et al., “Nano LC-MS/MS: a robust setup for proteomicanalysis” Methods Mol Biol. 790:115-126 (2011).

A preparation pathway for Nano-LC-MS/MS is outlined. See, FIG. 1. Ingeneral, an experimental design was followed that identified thepatient's age (e.g., young (Y), old (O)) in conjunction with thepresence or absence of sepsis (e.g., sepsis (Y), no sepsis (N). Oneexemplary experimental design is outlined. See, Table 1.

TABLE 1 Experimental design and iTRAQ quantitation channel assignmentreporter ion_114 reporter ion_115 reporter ion_116 reporter ion_1171^(st) Experiment YY^(a) YN^(b) OY^(c) ON^(d) 2^(nd) Experiment YY^(a)YN^(b) OY^(c) ON^(d) 3^(rd) Experiment YY^(a) YN^(b) OY^(c) ON^(d)4^(th) Experiment YY^(a) YN^(b) OY^(c) ON^(d) 5^(th) Experiment YN^(b)OY^(c) ON^(d) YY^(a) 6^(th) Experiment YN^(b) OY^(c) ON^(d) YY^(a)7^(th) Experiment YN^(b) OY^(c) ON^(d) YY^(a) 8^(th) Experiment OY^(c)ON^(d) YY^(a) YN^(b) 9^(th) Experiment OY^(c) ON^(d) YY^(a) YN^(b)10^(th) Experiment OY^(c) ON^(d) YY^(a) YN^(b) ^(a)subjects who are atthe age of 50-65 and with severe sepsis; ^(b)subjects who are at the ageof 50-65 and without severe sepsis; ^(c)subjects who are at the age of70-85 and with severe sepsis; ^(d)subjects who are at the age of 70-85and without severe sepsis.

One optimized workflow was applied to plasma derived from four groups ofsepsis patients. The four groups were: 1) 10 survivors at 60-65 yearswith severe sepsis, 2) 10 non-survivors at 60-65 years with severesepsis, 3) 10 survivors >80 years with severe sepsis and 4) 10non-survivors >80 years with severe sepsis. See, Table 2.

TABLE 2 Subject Profile For Determining Age-Related Outcomes In SepsisYounger Sepsis Patients Older Sepsis Patients Survivor Death SurvivorDeath (10 Subjects) (10 Subjects) (10 Subjects) (10 Subjects)Samples from the four groups were analyzed with the optimized workflowblindedly.

Chromatograms obtained from MARS depletion and tandem MARS-depletionwere shown to be reproducible (i.e., the relative standard deviation ofunbound peaks and bound peaks were 1.6% and 0.4%, respectively.). See,FIGS. 10A and 10B.

III. Sepsis Biomarker Identification

This invention includes the identification of more than eighty humanplasma proteins which may have the potential to serve as biomarkers forthe diagnosis of sepsis in elderly patients. See, Table 3.

TABLE 3 Protein Level Ratios Comparing Elderly Patients With and WithoutSevere Sepsis (OY/ON): Proteins were quantified in at least sixexperiments (≧1.30 or ≦0.77; Coefficient Variance ≦0.55) Acc No. ProteinName Mean SD P02741 C-reactive protein 0.521 0.221 P02671 Fibrinogenalpha chain 0.597 0.129 P02751 Fibronectin 0.624 0.314 P07996Thrombospondin-1 0.645 0.335 P02649 Apolipoprotein E 0.646 0.170 P01011Alpha-1-antichymotrypsin 0.683 0.183 Q9Y6R7 IgGFc-binding protein 0.7000.259 Q9BXR6 Complement factor H-related protein 5 0.716 0.181 Q86UD1Out at first protein homolog 0.742 0.193 P08779 Keratin, type Icytoskeletal 16 0.754 0.355 P02775 Platelet basic protein 0.758 0.211P18428 Lipopolysaccharide-binding protein 0.762 0.321 Q96PD5N-acetylmuramoyl-L-alanine amidase 1.347 0.393 Q15431 Synaptonemalcomplex protein I 1.372 0.620 O14791 Apolipoprotein L1 1.390 0.701P35858 Insulin-like growth factor-binding protein 1.393 0.462 complexacid labile subunit P19323 Inter-alpha-trypsin inhibitor heavy chain H21.397 0.528 P02656 Apolipoprotein C-III 1.426 0.769 O95445Apolipoprotein M 1.438 0.483 P02652 Apolipoprotein A-II 1.447 0.463P06276 Cholinesterase 1.452 0.474 P05090 Apolipoprotein D 1.470 0.321P19827 Inter-alpha-trypsin inhibitor heavy chain H1 1.576 0.582 P02749Beta-2-glycoprotein 1 1.582 0.611 P29622 Kallistatin 1.685 0.574 Q9NXD2Myotubularin-related protein 10 1.779 0.692 Q04756 Hepatocyte growthfactor activator 1.808 0.681 Q86UV6 Tripartite motif-containing protein74 1.847 0.737 Q6UXB8 Peptidase inhibitor 16 2.028 0.782 Q96KN2Beta-Ala-His dipeptidase 2.060 0.931 P02654 Apolipoprotein C-I 2.1651.022These proteins were identified and quantified using iTRAQ basedchemistry in combination with liquid chromatography and massspectrometry.

In one embodiment, the present invention contemplates a methodcomprising identifying protein pathway biomarkers of severe sepsis inpatients with an infection. In one embodiment, the present inventioncontemplates a method comprising developing biomarkers of severe sepsisin elderly patients with an infection. In one embodiment, the presentinvention contemplates a method comprising developing protein pathwaybiomarkers of severe sepsis in non-elderly patients with an infection.Although it is not necessary to understand the mechanism of an inventionit is believed that a sepsis proteomic pathway analysis may discoverpotential mechanisms and therapies for severe sepsis, potentialmechanisms and therapies for age-related immune dysfunction, and/orpotential mechanisms and therapies for age-related organ dysfunction insepsis.

The proteins identified in this study have not been implicated and/orpreviously identified in the plasma of various aged elderly severesepsis patients. For example, some interleukins were identified asdifferentially expressed. See, Table 4.

TABLE 4 Differential Interleukin Expression In Elderly Severe SepsisPatients Interleukin 21 YY/OY 0.626 ± 0.169 Interleukin 21 YN/ON 0.519 ±0.105 Interleukin 21 YY/YN 2.054 ± 0.105 Interleukin 21 OY/ON 1.735 ±0.443 Interleukin 19 YY/OY 0.508 ± 0.046 Interleukin 19 YN/ON 0.426 ±0.051 Interleukin 19 YY/YN 1.924 ± 0.185 Interleukin 19 OY/ON 1.609 ±0.108IL-21 is believed to be homologous to 11-2, TL-4 and IL-15 and isgenerally considered to be a proinflammatory cytokine. IL-19 is believedto belong to the IL-10 family of cytokines along with IL-10, IL-20,IL-22. IL-19 has been reported to be up-regulated in monocytes followingstimulation with LPS.

Some blood factors were also seen to be differentially expressed inelderly patients with severe sepsis. See, Table 5.

TABLE 5 Differential Blood Factor Expression In Elderly Patients WithSevere Sepsis YY/OY YN/ON YY/YN OY/ON Factor XIII, 0.710 ± 0.158 N.D.N.D. N.D. A Chain Factor XII 0.767 ± 0.093 N.D. N.D. 1.563 ± 1.163 Von1.161 ± 0.821 N.D. 2.047 ± 1.297 N.D. Willebrand Factor Fibrinogen 2.058± 0.577 0.246 ± 2.441 ± 0.719 0.597 ± 0.129 alpha 0.120 N.D. = NotDetermined

Some lipid factors were also seen to be differentially expressed iselderly patients with severe sepsis. See, Table 6.

TABLE 6 Differential Lipid Factor Expression In Elderly Patients WithSevere Sepsis YY/OY YY/ON YY/YN OY/ON Lipopolysaccharide 2.499 ± 0.604N.D. 2.201 ± 0.502 N.D. Binding Protein Apolipoprotein E 1.692 ± 0.3090.522 ± 0.164 2.105 ± 0.301 0.646 ± 0.170 Apolipoprotein AII 0.426 ±0.073 1.805 ± 0.685 0.502 ± 0.080 1.447 ± 0.463 Apolipoprotein 0.578 ±0.099 2.961 ± 1.372 0.690 ± 0.294 1.426 ± 0.769 CIIIThe data presented herein was derived from patients exhibiting symptomsof community acquired pneumonia using a semi-quantitative plasmaproteomics workflow. Cao et al., “Additions to the human plasma proteomevia a tandem MARS depletion iTRAQ-based workflow” International Journalof Proteomics 2013 Article ID 654356 ((2013). This technique includes,but is not limited to, tandem immunoaffinity depletion, isobaric tagsfor relative and absolute quantitation (iTRAQ) labeling, strong cationexchange (SCX) fractionation, and nanoflow liquid chromatography (LC)coupled to high resolution mass spectrometry (MS).

In one embodiment, the present invention contemplates a sepsis proteomicexpression profile comprising proteins that are differentially expressedbetween young adults without severe sepsis (YN), elderly adults withoutsevere sepsis (ON), young adults with severe sepsis (YY) and/or elderlyadults with severe sepsis (OY). Although it is not necessary tounderstand the mechanism of an invention, it is believed that manydifferentially-expressed proteins are unique to either younger or olderadults which may explain an increased risk of severe sepsis in olderadults. It is further believed that many differentially-expressedproteins that are common to the young and elderly adult groups may alsobe identified. The data presented herein demonstrate that thefold-change direction observed for differentially expressed proteins incommon between young and elderly patients varies depending on patientage and thus may explain higher risk of severe sepsis in older adults.

The data presented herein identify fifty-nine (59)differentially-expressed proteins were identified in comparisons fromboth age groups (i.e., YS/YC and OS/OC). See, Table 7.

TABLE 7 List of differentially-expressed proteins in patients withsevere sepsis as a function of age Acc. YS/YC OS/OC No.^(a) Protein Name(mean ± SD^(b)) (mean ± SD) O14791 Apolipoprotein L1 / 1.390 ± 0.701O95445 Apolipoprotein M / 1.438 ± 0.483 P01008 Antithrombin-III 0.596 ±0.064 / P01011 ^(c) Alpha-1-antichymotrypsin 1.577 ± 0.867 0.683 ± 0.183P02649 Apolipoprotein E 2.105 ± 0.301 0.646 ± 0.170 P02652Apolipoprotein A-II 0.502 ± 0.080 1.447 ± 0.463 P02654 ApolipoproteinC-I / 2.165 ± 1.022 P02656 Apolipoprotein C-III 0.690 ± 0.294 1.426 ±0.769 P02671 Fibrinogen alpha chain 2.441 ± 0.719 0.597 ± 0.129 P02675Fibrinogen beta chain 2.102 ± 0.579 / P02679 Fibrinogen gamma chain2.077 ± 0.720 / P02735 Serum amyloid A protein 3.058 ± 1.088 / P02741C-reactive protein 3.268 ± 1.070 0.521 ± 0.221 P02749Beta-2-glycoprotein 1 / 1.582 ± 0.611 P02750 Leucine-richalpha-2-glycoprotein 2.140 ± 0.296 / P02751 Fibronectin / 0.624 ± 0.314P02753 Retinol-binding protein 4 0.525 ± 0.088 / P02763 Alpha-1-acidglycoprotein 1 2.033 ± 0.426 / P02766 Transthyretin 0.440 ± 0.094 /P02774 Vitamin D-binding protein 0.568 ± 0.084 / P02775 Platelet basicprotein / 0.758 ± 0.211 P04114 Apolipoprotein B-100 1.523 ± 0.253 /P04259 Keratin, type H cytoskeletal 6B 0.521 ± 0.056 / P04275 vonWillebrand factor 2.047 ± 0.340 / P04278 Sex hormone-binding globulin0.698 ± 0.258 / P05090 Apolipoprotein D / 1.470 ± 0.321 P05154 Plasmaserine protease inhibitor 0.726 ± 0.457 / P05452 Tetranectin 0.640 ±0.103 / P05546 Heparin cofactor 2 0.707 ± 0.255 / P06276 Cholinesterase/ 1.452 ± 0.474 P06396 Gelsolin 0.546 ± 0.098 / P07996 Thrombospondin-1/ 0.645 ± 0.335 P08779 Keratin, type I cytoskeletal 16 / 0.754 ± 0.355P10909 Clusterin 0.731 ± 0.297 / P13645 Keratin, type I cytoskeletal 100.582 ± 0.064 / P15169 Carboxypeptidase N catalytic chain 1.312 ± 0.364/ P16070 CD44 antigen 1.618 ± 0.545 / P18428 Lipopolysaccharide-bindingprotein 2.201 ± 0.502 0.762 ± 0.321 P19652 Alpha-1-acid glycoprotein 22.169 ± 0.598 / P19823 Inter-alpha-trypsin inhibitor heavy chain / 1.397± 0.528 H2 P19827 Inter-alpha-trypsin inhibitor heavy chain / 1.576 ±0.582 H1 P20061 Transcobalamin-1 / / P20851 C4b-binding protein betachain 1.391 ± 0.460 / P29622 Kallistatin / 1.685 ± 0.574 P35542 Serumamyloid A-4 protein 0.476 ± 0.044 / P35858 Insulin-like growthfactor-binding protein / 1.393 ± 0.462 complex acid labile subunitP61626 Lysozyme C 1.905 ± 0.958 / P61769 Beta-2-microglobulin 1.887 ±1.020 / P80108 Phosphatidylinositol-glycan-specific 0.390 ± 0.064 /phospholipase D Q03591 Complement factor H-related protein 1 1.312 ±0.595 / Q04756 Hepatocyte growth factor activator / 1.808 ± 0.681 Q15431Synaptonemal complex protein 1 / 1.372 ± 0.620 Q6UXB8 Peptidaseinhibitor 16 / 2.028 ± 0.782 Q86UD1 Out at first protein homolog / 0.742± 0.193 Q86UV6 Tripartite motif-containing protein 74 / 1.847 ± 0.787Q96KN2 Beta-Ala-His dipeptidase / 2.060 ± 0.931 Q96PD5N-acetylmuramoyl-L-alanine amidase 0.576 ± 0.061 1.347 ± 0.393 Q9BXR6Complement factor H-related protein 5 / 0.716 ± 0.181 Q9NXD2Myotubularin-related protein 10 / 1.779 ± 0.692 Q9Y6R7 IgGFc-bindingprotein / 0.700 ± 0.259 ^(a)Accession number provided from theUniprothuman database(Apr. 25, 2010, 20295 sequences). ^(b)Mean and SDvalues are calculated based on the reporter ion ratios for proteinsquantified in at least 6 biological experiments. ^(c)Proteins quantifiedin both comparisons are in bold.The measured fold-change values reported in Table 7 include the mean andstandard deviation (SD) for each protein based on the ratios averagedacross all biological replicates.

As described herein, an iTRAQ-based semi-quantitative proteomicsworkflow was employed to identify differentially-expressed proteins in50-65 and 70-85 year old CAP patients who developed severe sepsis ascompared to those patients that did not. For example, a tandem MARSdepletion was performed on a MARS Hu-6 column to effectively remove highabundance proteins. The collected data was derived from patient samplesfrom the four groups (i.e., YC, YS, OC and OS) that were randomlyassigned to one of four iTRAQ reagents in a blind fashion across tenSCX-LC-MS/MS experiments.

An exemplary LC chromatogram for 13 SCX fractions of pooled iTRAQ 4-plexsamples is shown. See, FIG. 22. Triplicate LC-MS/MS runs performed foreach fraction were reproducible. Example spectra demonstrate peakisolation and fragmentation. See, FIGS. 22B-G. The data showdoubly-charged peptides at m/z 748.9046 and 742.9010 in SCX fractions 7and 6 that were eluted from the column at t_(r)=57.20 min and 37.26 min,respectively. See, FIGS. 22B and 22C, respectively. The CID MS/MSspectra display a consecutive series of b- and y-fragment ions used toassign the peptides as [YYTYLIMNK+2H]²⁺ of protein Complement C3 and[GWVTDGFSSLK+2H]²⁺ of protein Apolipoprotein CIII, respectively. See,FIGS. 22D and 22E, respectively. A mass increase of 145 Da was observedfor b1 and y1 ions for each of the two peptides, indicating iTRAQlabeling at the N-terminus and the presence of lysine at the C-terminus.HCD MS/MS spectra also contain a series of b- and y-ions used to confirmthe sequence of peptides. See, FIGS. 22F and 22G, respectively. Thelower m/z region of the HCD MS/MS spectra shows the intensity of thereporter ions which is used to quantify the peptides. See, inserts,FIGS. 22F and 22G, respectively. The ratios across the four groups were1.0:0.9:1.0:1.0 and 1.0:1.3:2.5:0.5 for reporter ions 114:115:116:117from Complement C3 and Apo CIII, respectively. See, FIGS. 22F and 22G,respectively.

The data presented herein shows an average number±standard deviation ofspectral counts (SCs) and proteins identified in each of the tenbiological replicate experiments. See, FIG. 23. An average of 283±14proteins and 59,645±4129 spectral counts were identified and similarresults were obtained across individual experiments. The total number ofproteins identified increases with each pooled sample experiment suchthat a total of 772 unique proteins were identified from all plasmasamples. Based on SCs, the three most abundant proteins are ComplementC3 (49,749 SCs), α-2-macroglobulin (47,273 SCs) and Apolipoprotein A1(28,261 SCs).

Western blotting analysis was employed to generate a secondarymeasurement for several differentially expressed proteins. Proteinsinvolved in an acute phase response (i.e., CRP), coagulation pathway(i.e., FAC) pathway and lipid metabolism (i.e., ApoCIII) were selected.See, FIGS. 24A-C. The histogram plots represent the normalizedintensities corresponding to the density of the band spots for eachgroup. The relative abundance obtained from the iTRAQ-based sepsisproteomics workflow of CRP, FAC and ApoCIII were 3.27:1.00:1.74:3.34,2.44:1.00:2.97:4.98, and 0.69:1.00:0.58:0.40, respectively.

These observed Western blot data are consistent with the measured iTRAQresults for the fifty-nine differentially-expressed proteins that wereidentified in comparisons from both age groups (i.e., YS/YC andOS/OC)(Y=young, O=elderly; S=sepsis, C=control or without sepsis). See,Table 7. The measured fold-change values reported in the table includethe mean and SD for each protein based on the ratios averaged across allbiological replicates. As shown, 28 proteins aredifferentially-expressed in the population of 50-65 year olds (i.e.,YS/YC) in which 14 have higher levels and 14 have lower levels inpatients with severe sepsis compared to those with CAP. See, FIG. 25. Inthe population of 70-85 year olds (i.e., OS/OC), 23 proteins aredifferentially-expressed, in which 16 and seven have higher and lowerlevels in patients with severe sepsis, respectively. Of the 59 totaldifferentially-expressed proteins, eight are in common amongst both agegroups. Interestingly, however the direction of the fold change differsin younger and older adults. Specifically, alpha-1-antichymotrypsin(A1ACT), apolipoprotein (Apo) E, fibrinogen α chain, C-reactive protein(CRP), and LPS binding protein (LBP) levels were higher in youngeradults with severe sepsis but lower in older adults with severe sepsisrelative to age-matched controls, suggesting that lower levels of theseproteins is associated with increased risk and incidence of severesepsis in older adults. On the other hand, Apo AII, Apo CIII, andN-acetylmuranoyl-L-anlanineamidase have lower and higher levels inyounger and older with severe sepsis, respectively, suggesting thathigher levels of these proteins may be factors for increased severesepsis risk in older adults.

Using an Ingenuity Pathway Analysis method, nineteen (19) biologicalpathways were significantly over-represented (p<0.05) for the 59identified differentially-expressed proteins. See, FIG. 26. The mostrepresented pathways include LXR/RXR activation and acute phase responsesignaling whereby 18 proteins are associated with each of thesepathways. Next, many differentially-expressed proteins are involved inatherosclerosis signaling, interleukin (IL)-12 signaling and productionof NO and reactive oxygen species in macrophages, and endocytosissignaling. Fewer proteins are involved in the remaining biologicalpathways such as actin cytoskeleton and IL-6 signaling.

IV. Sepsis Proteomic Pathway Expression Profiles

In one embodiment, the present invention contemplates determining theeffects of aging on severe sepsis risk by evaluating, an acute plasmaproteome. While sepsis can occur as early as the neonatal stage,incidence and morbidity increases with age and rises sharply after 65years, presumably due to immunosenescence and high levels ofinflammatory proteins. Martin et al., “The effect of age on thedevelopment and outcome of adult sepsis” Critical Care Medicine 34:15-21(2006); and Opal et al., “The immunopathogenesis of sepsis in elderlypatients” Clinical Infectious Diseases 41:S504-S512 ((2005); McDonald etal., “Aging is associated with impaired thrombus resolution in a mousemodel of stasis induced thrombosis” Thrombosis Research 125:72-78(2010); Turnbull et al., “Effects of aging on the immunopathologicresponse to sepsis. Critical Care Medicine 37, 1018-1023 (2009);Yamamoto et al., “Aging accelerates endotoxin-induced thrombosis:increased responses of plasminogen activator inhibitor-1 andlipopolysaccharide signaling with aging” The American Journal ofPathology 161:1805-1814 (2002); Cohen et al., “Coagulation andactivation of inflammatory pathways in the development of functionaldecline and mortality in the elderly” The American Journal of Medicine114:180-187 (2003); Downie et al., “Community-acquired neonatal andinfant sepsis in developing countries: efficacy of WHO's currentlyrecommended antibiotics-systematic review and meta-analysis” Archives ofDisease in Childhood 98:146-154 (2013); Karumbi et al., “Topicalumbilical cord care for prevention of infection and neonatal mortality”Pediatric Infectious Disease Journal 32:78-83 (2013); Kaplan et al.,“Hospitalized community-acquired pneumonia in the elderly” AmericanJournal of Respiratory and Critical Care Medicine 165:766-772 (2002);Girard et al., “Bacteremia and sepsis in older adults” Clinics InGeriatric Medicine 23:633-647 (2007); Fulop et al., “Role ofimmunosenescence in infections and sepsis in the elderly” In: Handbookon Immunosenescence, Fulop et al., Eds., pp. 965-977, (2009) SpringerNetherlands; and Bruunsgaard et al., “Impaired production ofproinflammatory cytokines in response to lipopolysaccharide (LPS)stimulation in elderly humans” Clinical And Experimental Immunology118:235-241 (1999). However, none of these reports have identifiedsignificant differences with age for inflammatory and coagulationproteins and cell surface markers in patients with severe sepsis. Kaleet al., “The effects of age on inflammatory and coagulation-fibrinolysisresponse in patients hospitalized for pneumonia” PLoS ONE 5:e13852(2010).

The data presented herein provide the basis for an age-related sepsisproteomic expression profile that identified proteins involved variousbiological pathways including, but not limited to, acute phasesignaling, coagulation pathway, and lipid/or metabolism that aredifferentially-expressed in an elderly population.

A. Acute Phase Response Pathways

Sepsis is believed to induce a pro-inflammatory state which can becharacterized by elevated levels of pro-inflammatory cytokines, such asIL-6, IL-10 and tumor necrosis factor (TNF)-α. Cohen J., “Theimmunopathogenesis of sepsis” Nature 420:885-891 (2002). Thesepro-inflammatory cytokines regulate acute phase responses and theexpression of acute phase proteins (APPs). Altered expression of APPshas been demonstrated in sepsis. For example, increased levels ofα-1-acid glycoprotein (A1AG), A1ACT, and LBP and decreased levels oftransthyretin (TTR) are observed in 65 year old sepsis patients andcecal ligand/puncture (CLP) mouse and pig models. It also appears thatacute phase response increases with the disease severity. For example,sepsis patients have higher levels of CRP and lower levels of serumamyloid A4 relative to patients with systemic inflammatory responsesyndrome (SIRS), another precursor to sepsis. Póvoa et al., “C-reactiveprotein: a valuable marker of sepsis” Intensive Care Medicine 28:235-243(2002); Shen et al., “Sepsis Plasma Protein Profiling withImmunodepletion, Three-Dimensional Liquid Chromatography Tandem MassSpectrometry, and Spectrum Counting” Journal of Proteome Research5:3154-3160 (2006); Zweigner et al., “High concentrations oflipopolysaccharide-binding protein in serum of patients with severesepsis or septic shock inhibit the lipopolysaccharide response in humanmonocytes” Blood 98:3800-3808 (2001); Kalenka et al., “Changes in theserum proteome of patients with sepsis and septic shock” Anesthesia &Analgesia 103:1522-1526 (2006); Ren et al., “The alterations of mouseplasma proteins during septic development” Journal of Proteome Research6:2812-2821 (2007); Thongboonkerd et al., “Altered plasma proteomeduring an early phase of peritonitis-induced sepsis” Clinical Science116:721-730 (2009).

In the data presented herein it was observed that eighteen (18)differentially-expressed proteins including, but not limited to, CRP,LBP, A1ACT, and TTR may be involved in acute phase response and thelevels in younger adults are consistent with above cited studies. See,FIG. 26. For example, CRP (fold-change YS/YC 3.268±1.070), LBP(2.201±0.502), A1ACT (1.577±0.867), and A1AG (2.033±0.426) have higherconcentrations while TTR (0.440±0.094) has lower concentrations inyounger adults who developed severe sepsis as compared to those did not.Also evidenced in the present data is that acute phase response alsovaries depending on patient age. The proteins CRP (0.521±0.221), LBP(0.762±0.321), and A1ACT (0.683±0.183) have lower concentrations inolder adults who later developed severe sepsis suggesting that lowerexpression of these proteins at older age leads to severe sepsis. CRPand LBP have protective effects by neutralizing the toxicity ofpathogens, such that decreased expression of these proteins in olderpatients may help explain higher risk and mortality of severe sepsis.Póvoa et al., “C-reactive protein as a marker of infection in criticallyill patients” Clinical Microbiology and Infection 11:101-108 (2005);Masia et al., “Serum concentrations of lipopolysaccharide-bindingprotein as a biochemical marker to differentiate microbial etiology inpatients with community-acquired pneumonia” Clinical Chemistry50:1661-1664 (2004); Tschaikowsky et al., “Lipopolysaccharide-bindingprotein for monitoring of postoperative sepsis: complemental toC-reactive protein or redundant?” PLoS ONE 6:e23615 (2011); and Villaret al., “Serum lipopolysaccharide binding protein levels predictseverity of lung injury and mortality in patients with severe sepsis”PLoS ONE 4:e6818 (2009). Both aging and severe sepsis alter the immunesystem and response to infection. Sadeghi et al., “Phenotypic andfunctional characteristics of circulating monocytes of elderly persons”Experimental Gerontology 34:959-970. Taken together, these results ofacute phase response imply that a hypoinflammatory response in olderadults may increase risk of severe sepsis, consistent with otherliterature reports. Welty-Wolf et al., “Proinflammatory cytokinesincrease in sepsis after anti-adhesion molecule therapy” Shock13:404-409 (2000); and Gustot T., “Multiple organ failure in sepsis:prognosis and role of systemic inflammatory response” Curr. Opin. Crit.Care 17:153-159 (2011).

B. Blood Factor Coagulation Pathways

Pro-inflammatory cytokines can also activate the coagulation pathway.Briefly, the activation of a series of coagulation factors cleaveprothrombin to thrombin, which in turn converts soluble fibrinogen intofibrin, upon which cross-linked fibrin forms blood clots. In sepsispatients, pro-coagulation overwhelms fibrinolysis (or anti-coagulation)leading to the presence of more blood clots. This has been demonstratedby decreased levels of anti-coagulant proteins (e.g., antithrombin III(ATIII), and heparin cofactor II) and increased levels of pro-coagulantproteins (e.g., fibrinogen, Von Willebrand factor (VWF)) in sepsispatients. Amaral et al., “Coagulation in sepsis” Intensive Care Medicine30:1032-1040 (2004); Soares et al., “Differential proteomics of theplasma of individuals with sepsis caused by Acinetobacter baumannii”Journal of Proteomics 73:267-278 (2009); Ware et al., “Significance ofvon Willebrand factor in septic and nonseptic patients with acute lunginjury” American Journal of Respiratory and Critical Care Medicine170:766-772 (2004); and Kaspereit et al., “The effect of fibrinogenconcentrate administration on coagulation abnormalities in a rat sepsismodel” Blood Coagulation & Fibrinolysis 15:39-43 (2004).

In the presently disclosed data, elevated levels of fibrinogen alphachain (2.441±0.579), fibrinogen beta chain (2.102±0.579), fibrinogengamma chain (2.077±0.720) and VWF (2.047±0.340) were found in youngeradults who developed severe sepsis. Fibrinogen has higher concentrationsand ATIII has lower concentrations in sepsis. Ren et al., “Thealterations of mouse plasma proteins during septic development” Journalof Proteome Research 6:2812-2821 (2007); and Soares et al.,“Differential proteomics of the plasma of individuals with sepsis causedby Acinetobacter baumannii” Journal of Proteomics 73:267-278 (2009).

Higher levels of VWF are associated with mortality in sepsis, whilehigher levels of fibrinogen and VWF may indicate the presence of moreblood clots in younger patients, consistent with literature reports,whom developed severe sepsis. Ware et al., “Significance of vonWillebrand factor in septic and nonseptic patients with acute lunginjury” American Journal of Respiratory and Critical Care Medicine170:766-772 (2004); Kaspereit et al., “The effect of fibrinogenconcentrate administration on coagulation abnormalities in a rat sepsismodel” Blood Coagulation & Fibrinolysis 15:39-43 (2004); van′t Veer etal., “Keeping blood clots at bay in sepsis” Nat Med 14:606-608 (2008);and Sunder-Plassmann et al., “Disseminated intravascular coagulation anddecrease in fibrinogen levels induced by vincristine/prednisolonetherapy of lymphoid blast crisis of chronic myeloid leukemia” Ann.Hematol. 62:169-173 (1991). This notion is further supported bydecreased concentrations of ATIII (0.596±0.064) and heparin cofactor II(0.707±0.255) in these studies. Interestingly, fibrinogen (0.597±0.129)had lower levels in older adults who developed severe sepsis. Althoughit is not necessary to understand the mechanism of an invention, it isbelieved that the presently disclosed proteomic sepsis expressionprofile indicates that an acute response of reduced coagulation activitymay be a contributing factor to higher incidence and mortality of severesepsis found in older adults.

C. Lipid Metabolism Pathways

Apolipoproteins have been suggested to play a role in liver Xreceptor/retinoid X receptor (LXR/RXR) activation and atherosclerosissignaling. For example, activated LXR/RXR complex can enhance theprocess of reverse cholesterol transport and cholesterol efflux, whichtransfers accumulated cholesterol from the blood vessel walls to theliver for excretion. Larrede et al., “Stimulation of cholesterol effluxby LXR agonists in cholesterol-loaded human macrophages isABCA1-dependent but ABCG1-independent” Arteriosclerosis, Thrombosis, andVascular Biology 29:1930-1936 (2009); and Zhao et al., “Liver X receptorin cholesterol metabolism” Journal of Endocrinology 204:233-240 (2010).Additionally, activated LXR/RXR can reduce the expression ofpro-inflammatory cytokines (e.g., IL-6, IL-1β) by inhibition of theactivity of transcription factor NF-κB. Myhre et al., “Liver X receptoris a key regulator of cytokine release in human monocytes” Shock29:468-474 (2008). Interestingly, acute phase response pathway proteins,which are activated during aging and severe sepsis (supra), can inhibitLXR/RXR activation. Khovidhunkit et al., “Thematic review series: thepathogenesis of atherosclerosis. effects of infection and inflammationon lipid and lipoprotein metabolism mechanisms and consequences to thehost” Journal of Lipid Research 45:1169-1196 (2004). Failed activationof the LXR/RXR complex may lead to atherosclerosis, which is associatedwith severe sepsis Brazil, M., “Macrophage LXRs inhibit atherosclerosis”Nat Rev Drug Discov 1:840-840 (2002); Joseph et al., “Synthetic LXRligand inhibits the development of atherosclerosis in mice” Proceedingsof the National Academy of Sciences 99:7604-7609 (2002); Hackam et al.,“Statins and sepsis in patients with cardiovascular disease: apopulation-based cohort analysis” Lancet 367:413-418 (2006); and Podnoset al., “Intra-abdominal Sepsis in Elderly Persons” Clinical InfectiousDiseases 35:62-68 (2002).

Altered levels of apoliproteins have been reported in sepsis, Forexample, elevated levels of Apo B-100 and lower concentrations of ApoCIII in response to inflammation. Phetteplace et al., “Escherichia colisepsis increases hepatic apolipoprotein B secretion by inhibitingdegradation” 35:1079-1085 (2000); and Lacorte et al., “Repression ofapoC-III gene expression by TNFα involves C/EBPδ/NF-IL6β via an IL-1independent pathway” FEBS Letters 415:217-220 (1997). The data providedherein also shows an increased expression of Apo B-100 (1.523-0.253) anddecreased expression of Apo CIII (0.690±0.294) in younger adults whodeveloped severe sepsis. Although it is not necessary to understand themechanism of an invention, it is believed that these changes may suggestsuppressed activation of the LXR/RXR pathway and subsequent higher riskof atherosclerosis in younger adults. Higher levels of Apo E(2.105±0.301) were found in younger adults, however lower levels(0.646±0.170) were detected in older adults. Kattan et al.,“Apolipoprotein E-mediated immune regulation in sepsis” J Immunol181:1399-1408 (2008). Also, higher levels of Apo CI (2.165±1.022) andlower levels of Apo M (1.438±0.483) were detected in older adults whodeveloped severe sepsis. Apo CI levels correlate with survivorship in˜25 year old severe sepsis patients and may promote atherosclerosis.Berbée et al., “Plasma apolipoprotein CI correlates with increasedsurvival in patients with severe sepsis” Intensive Care Medicine34:907-911 (2008); Westerterp et al., “Apolipoprotein CI aggravatesatherosclerosis development in ApoE-knockout mice despite mediatingcholesterol efflux from macrophages” Atherosclerosis 195:e9-e16 (2007);and Westerterp et al., “Apolipoprotein C-I is crucially involved inlipopolysaccharide-induced atherosclerosis development in apolipoproteinE-knockout mice” Circulation 116:2173-2181 (2007). Apo M has also beenreported to have higher concentrations in severe sepsis patients andcontributes to the anti-inflammatory response in sepsis. Christoffersenet al., “Apolipoprotein M—a new biomarker in sepsis” Critical Care16:126 (2012); and Kumaraswamy et al., “Decreased plasma concentrationsof apolipoprotein M in sepsis and systemic inflammatory responsesyndromes” Crit Care 16:R60 (2012). Although it is not necessary tounderstand the mechanism of an invention, it is believed that higherlevels of Apo M in older adults who developed severe sepsis suggest thatthese patients have a reduced inflammatory state during acute response.It is further believed that lipid metabolism is altered in these studiespoints to atherosclerosis as a contributing factor of severe sepsisincidence.

D. Interleukin (IL) Pathways

Pathways involved in 1-12 and 11-6 signaling were identified in theproteomic sepsis expression provide that support observations of theirinvolvement in hypoinflammatory response in older adults. See, Table 7.Diefenbach et al., “Requirement for type 2 NO synthase for IL-12signaling in innate immunity:” Science 284:951-955 (1999).

E. Nitric Oxide/Reactive Oxygen Species Pathways

Pathways of differentially-expressed proteins were identified thatinvolve the production of nitric oxide (NO) and reactive oxygen species(ROS) (i.e., for example, retinol binding protein 1, lysozyme C and/orclusterin). Enhanced production of nitric oxide (NO) and reactive oxygenspecies (ROS) in macrophages has been previously reported in aging andsepsis which support elevated oxidative stress in the elderly and severesepsis patients. Kolls J. K., “Oxidative stress in sepsis: a redoxredux” The Journal of Clinical Investigation 116:860-863 (2006); Berr,C., “Cognitive impairment and oxidative stress in the elderly: Resultsof epidemiological studies” BioFactors 13:205-209 (2000); Karolkiewiczet al., “Oxidative stress and antioxidant defense system in healthy,elderly men: relationship to physical activity” The Aging Male 6:100-105(2003); and Macdonald et al., “Oxidative stress and gene expression insepsis” British Journal of Anaesthesia 90:221-232 (2003).

In one embodiment, the present invention contemplates measuringimmune-related oxidative stress. In one embodiment, the presentinvention contemplates determining the effects of adriamycin treatmenton the proteome. In one embodiment, the present invention contemplatesestablishing quantitative plasma workflow for age-related sepsisoutcomes.

F. Retinoid X Receptor Pathways

Different biological pathways have been identified to be associated withthe age-related risk of severe sepsis among CAP patients (supra). Amongthese, four pathways related to retinoid X receptor (RXR) areidentified: liver X receptor (LXR)/RXR activation, farnesoid X receptor(FXR)/RXR activation, LPS/IL-1 mediated inhibition of RXR function, andperoxisome proliferator-activated receptor alpha (PPARα)/RXRαactivation.

RXR has been reported to play a role in the nuclear receptor superfamilybecause it forms heterodimers with many other nuclear receptors andtherefore are involved in a variety of physiological processes includingimmune response. Mangelsdorf et al., Cell 83:841 (1995). For example, ininnate immune systems, activation of RXR has been shown to prevent theapoptosis and inflammatory response of macrophages. Núñez et al.,Proceedings of the National Academy of Sciences (2010). In the adaptiveimmune system, RXR has been reported to regulate the differentiation ofT-cells into regulatory or helper T-cells. Takeuchi et al., “The Journalof Immunology” 191:3725 (2013). Knockout of RXR gene has been reportedto result in diminished proliferation and increased apoptosis inT-cells. Stephensen et al., Immunology 121:484 (2007). In addition, RXRhas been reported to protect human endothelial cells from oxidativestress induced by high-dose glucose. Chai et al., Free Radical Biologyand Medicine 44:1334 (2008).

In sepsis, infection-induced damage to liver has been reported to bereduced by the activation of RXR. Chen et al., Shock 28:65 (2007).However, the effects of RXR on T-cells in septic infection have notexamined yet. Although it is not necessary to understand the mechanismof an invention, it is believed that activation of RXR may protectT-cells from oxidative stress induced by infection (e.g., LPSstimulation). To test this hypothesis, CD4+ T-cells were isolated fromthe spleen harvested from wild type mice at 6-month old. These cellswere cultured in the cell medium with or without LPS-stimulation for 24hours. LPS-stimulated CD4+ T-cells have increased levels of oxidativestress compared to the controls. Increased levels of RXR are alsoobserved in LPS-stimulated CD4+ T-cells (data not shown). These findingssuggest that increased levels of RXR might be responsible for theincreased protein expression induced by LPS in CD4+ T-cells. Forexample, gene for Apo E is one of the targets for RXR. Sureda et al.,Current Pharmaceutical Design 17:230 (2011). The data presented hereinshowing an increased plasma concentration of ApoE in young severe sepsispatients (e.g., 55-65 years old) is consistent with the abovesuggestion, as increased ApoE may be mediated by RXR activation.

IV. Sepsis Treatment: Current State of the Art

Conventional sepsis treatment is generally supportive usually requiringintravenous antibiotic administration in a hospital or other clinicalsetting. Further, oxygen administration with intravenous fluidreplacement is common. Secondary treatment may include, but are notlimited to, medications that increase blood pressure, dialysis and/ormechanical ventilation. Although it is not necessary to understand themechanism of an invention, it is believed that the data disclosed hereinsuggest that effective new sepsis therapies may be directed atalleviating the phenomenon of immunosensecence.

A broad-spectrum antimicrobial treatment is usually recommended forsepsis therapy that is initiated as soon as possible after diagnosis.This is because early and adequate antimicrobial therapy has beenreported to reduced mortality rates. American Thoracic Society,“Infectious Diseases Society of America. Guidelines for the managementof adults with hospital-acquired, ventilator-associated, andhealthcare-associated pneumonia” American Journal Respiratory andCritical Care Medicine 171(4):388-416 (2005); Harbarth et al.,“Inappropriate initial antimicrobial therapy and its effect on survivalin a clinical trial of immunomodulating therapy for severe sepsis”American Journal of Medicine 115(7):529-535 (2003); Kumar et al.,“Duration of hypotension before initiation of effective antimicrobialtherapy is the critical determinant of survival in human septic shock”Critical Care Medicine 34(6):1589-1596 (2006); Micek et al.,“Pseudomonas aeruginosa bloodstream infection: importance of appropriateinitial antimicrobial treatment” Antimicrobial Agents Chemotherapy49(4):1306-1311 (2005); and Proulx et al., “Delays in the administrationof antibiotics are associated with mortality from adult acute bacterialmeningitis” Quarterly Journal of Medicine 98(4):291-298 (2005).Unfortunately this approach can expose individuals to an overuse ofantimicrobials. This is mainly because of emerging resistant pathogens,which increase the risk of inappropriate therapy. Leone et al.,“Ventilator-associated pneumonia: Breaking the vicious circle ofantibiotic overuse” Critical Care Medicine 35(2):379-385 (2007); andNiederman et al., De-escalation therapy in ventilator-associatedpneumonia. Current Opinion in Critical Care 12(5):425-427 (2006).

Standard antimicrobial therapy comprises the maintenance of an initialempirical broad-spectrum antimicrobial therapy independent of whetherthe antimicrobial therapy was a combination or a single agent.Alternatively, de-escalation antimicrobial therapy comprises changing aninitially appropriate antimicrobial therapy from an empiricalbroad-spectrum characteristic to a narrower-spectrum one by eitherchanging the antimicrobial agent or by discontinuing an eventualantimicrobial combination, or both, according to culture results orclinical conditions.

In one embodiment, the present invention contemplates a method fortreating a sepsis patient using a therapy including, but not limited to,fluid therapy, vasopressor therapy, inotropic support, antibiotics,albumin and red blood cell transfusion in patients with sepsis. In oneembodiment, the vasopressor comprises norepinephrine and/or dopamine.Ibsen et al., “Perioperative treatment of patients with sepsis” CurrOpin Anaesthesiol. 26(3):348-353 (2013).

As discussed above, the clinical process of severe sepsis may becharacterized by extreme inflammation interlinked with potentstimulation of the coagulation cascade often followed by a state ofrelative immune paralysis. In some embodiments, the present inventioncontemplates treating a patient with sepsis with a therapy directed atleast one inflammatory cascade step. In one embodiment, the cascade stepcomprises modulating inflammatory mediators eliciting the immuneresponse. In one embodiment, the cascade step comprises altering thehost's immune response in both a stimulatory and depressive manner. Inone embodiment, the cascade step comprises taming an overexuberantcoagulation response triggered by a coagulation-inflammation cycle.Bernard et al., “The Immune Response: Targets for the Treatment ofSevere Sepsis” Int J Inflam., epub 2012:697592 (2012).

A. Antibiotic Therapy

In one embodiment, the present invention contemplates a method oftreating a sepsis patient by the administration of a broad-spectrumantibiotic. In one embodiment, a broad spectrum antibiotic includes, butis not limited to, ampicillin, amoxicillin, carbapenems, imipenem,meropenem, ertapenem, piperacillin, tazobactam, levofloxacin,gatifloxacin, moxifloxacin, ciprofloxacin, streptomycin, tetracycline,chloramphenicol and/or ticarcillin. Although it is not necessary tounderstand the mechanism of an invention a broad-spectrum antibiotic isbelieved to be an antibiotic that acts against a wide range ofdisease-causing bacteria. For example, a broad-spectrum antibiotic actsagainst both Gram-positive and Gram-negative bacteria, in contrast to anarrow-spectrum antibiotic, which is effective against specific familiesof bacteria.

Early, appropriate antibiotic therapy is usually recommended for thetreatment of the septicemic patient. The degree of organ dysfunction,underlying medical conditions, and physiologic abnormalities areprognostic factors but are not necessarily prognostic for initialantibiotic selection. Initial empiric therapy should be directed againstthe resident flora of the organ, which is primarily involved in theinfectious process. Blood cultures should be obtained in all patientsfor the initiation of antibiotic therapy, and methods should be employedfor the early detection of septicemia. Other conditions that mimicsepsis, e.g., pseudosepsis, should be ruled out initially to avoid anincorrect diagnosis and unnecessary antibiotic therapy. Monotherapy andfully recommended doses of antimicrobial drugs delivered by theintravenous route as soon as the diagnosis is established remain thecornerstone of therapy in treating the septic patient. Monotherapy withan antibiotic of the appropriate spectrum is more than adequate to treatthe great majority of septicemic patients. Double-drug therapy isrecommended to treat febrile leukopenic compromised hosts, serious P.aeruginosa infections, and selected cases of intra-abdominal sepsis.Cunha B A., “Antibiotic treatment of sepsis” Med Clin North Am. 1995May; 79(3):551-558.

Antibiotic selection made within the first hour of recognition of severesepsis and septic shock has been shown to decrease mortality. Methods ofdetermining what antibiotics should be prescribed and identification offactors influencing ineffective antibiotic coverage in patients withsevere sepsis or septic shock have been reported. For example, aretrospective review of emergency department (ED) patients admitted toan intensive care unit (ICU) over a 12-month period with aculture-positive diagnosis of either severe sepsis or septic shock wasperformed. Appropriate antibiotic therapy was defined as effectivecoverage of the offending organism based on final culture results. Ofthe 1400 patients admitted to the ICU, 137 patients were culturepositive and met the criteria for severe sepsis or septic shock.Effective antibiotic coverage was prescribed by emergency physicians in82% (95% confidence interval [CI]0.74-0.88) of cases. Of the 25 patientswho received ineffective antibiotics, the majority had infections causedby resistant Gram-negative organisms. Health care-associated pneumoniaguidelines were applied to all patients, regardless of the source ofinfection, and were 100% sensitive (95% CI 0.93-1.0) for selectingpatients who had infections caused by highly resistant organisms.Emergency physicians achieved 82% effective antibiotic coverage inpatients with severe sepsis or septic shock. The gap seems to be incoverage of highly resistant Gram-negative organisms. An alternativeapproach to antibiotic prescription, utilizing a set of guidelines forcommunity- and health care-associated infections, was found to be 100%sensitive in selecting patients who had infections caused by the moreresistant organisms. Capp et al., “Effective antibiotic treatmentprescribed by emergency physicians in patients admitted to the intensivecare unit with severe sepsis or septic shock: where is the gap?” J EmergMed. 2011 41(6):573-580.

Antibiotics are generally used to treat bacterial sepsis because it isbelieved that they reduce the bacterial burden. The impact of bacterialresistance has recently been studied and found to be important in arange of conditions. Resistance to antibiotics can be definedgenotypically, phenotypically and clinically throughpharmacokinetic/pharmacodynamic studies and their correlations withclinical outcomes. Although the kinetics of antibiotics has been shownto be favourably altered in sepsis, a range of studies in sepsis hasrevealed that for most pathogens resistance contributes to significantincreases in mortality. This has been demonstrated in bacteraemia,including community- and hospital-acquired infection, and withbacteraemia caused by vancomycin-resistant enterococci,methicillin-resistant staphylococci and extended-spectrum producingGram-negative bacteria. Significant mortality increases have also beenseen with ventilator-associated pneumonia and serious infectionsrequiring admission to intensive care. Gentotypic and phenotypicresistance in coagulase-negative staphylococci causing bacteraemia, andin invasive pneumococcal disease has not shown differences in mortality.In the latter case, dosage regimens have to date been adequate toovercome laboratory-defined resistance. Early indications are thatde-escalating therapy from broad-spectrum initial coverage after resultsof cultures and susceptibility tests become available does notjeopardize outcomes, and further prospective studies are warranted.There is now convincing evidence that broad-spectrum initial therapy tocover the likely pathogens and their resistances pending culture resultsis useful in sepsis to minimize adverse outcomes. Turnidge J., “Impactof antibiotic resistance on the treatment of sepsis” Scand J Infect Dis.2003; 35(9):677-682.

B. Inflammatory Mediator Inhibitor Therapy

Decades ago, unfruitful attempts were made to create antibodies with thepotential to bind and to prevent inflammatory bacterial components fromtriggering the hyperinflammatory response of sepsis. Lipopolysaccharide(LPS), a primary mediator in gram-negative sepsis, was the target ofresearchers as early as the 1980s. Clinicians tested E5 and HA1A, bothanti-LPS monoclonal antibodies, as treatments for septic patients. Ininitial studies, both antibodies showed encouraging results in smallsubsets of patients. Fink showed improvement in mortality in patientswith culture-proven gram-negative bacteremia when treated with HA1A.Fink M P. “Adoptive immunotherapy of gram-negative sepsis: use ofmonoclonal antibodies to lipopolysaccharide” Critical Care Medicine21(supplement 2):S32-S39 (1993). Ziegler et al. showed improvedmortality with the use of HA-1A therapy in 200 patients with provengram-negative sepsis. The 343 septic patients without culture provengram-negative bacteremia showed no treatment benefit. Ziegler et al.,“Treatment of gram-negative bacteremia and septic shock with HA-1A humanmonoclonal antibody against endotoxin. A randomized, double-blind,placebo-controlled trial” New England Journal of Medicine. 1991;324(7):429-436. Greenman et al. evaluated E5 in 1991 and showed improvedmortality and resolution of organ failure in a subgroup of patients notin shock at the time of study entry. Greenman et al., “A controlledclinical trial of E5 murine monoclonal IgM antibody to endotoxin in thetreatment of gram-negative sepsis” Journal of the American MedicalAssociation. 1991; 266(8):1097-1102. In a follow-up study, Bone et al.evaluated 530 patients with suspected or proven gram-negative sepsis anddid not find a difference in mortality but demonstrated improvement oforgan failure resolution in those treated with E5 as well as preventionof adult respiratory distress syndrome and central nervous system organfailure. Bone et al., “A second large controlled clinical study of E5, amonoclonal antibody to endotoxin: results of a prospective, multicenter,randomized, controlled trial” Critical Care Medicine. 1995;23(6):994-1006. Unfortunately, further studies of these therapies inlarger clinical trials including more than 1,000 patients each wereunable to confirm efficacy. Alejandria et al., “Intravenousimmunoglobulin for treating sepsis and septic shock” Cochrane Databaseof Systematic Reviews. 2002; (1):p.CD001090; Angus et al., “E5 murinemonoclonal antiendotoxin antibody in gram-negative sepsis: a randomizedcontrolled trial” Journal of the American Medical Association. 2000;283(13): 1723-1730; McCloskey et al., “Treatment of septic shock withhuman monoclonal antibody HA-1A: a randomized, double-blind,placebo-controlled trial” Annals of Internal Medicine. 1994; 121(1):1-5.

More recently, this approach has been revisited with the concept ofinhibiting toll-like receptor 4 (TLR-4) which is expressed on thesurface of immune cells and binds LPS and other ligands to initiate anintracellular signaling cascade resulting in the release ofproinflammatory cytokines. Salomao et al., “TLR signaling pathway inpatients with sepsis” Shock. 2008; 30(supplement 1):73-77. The therapy,TAK-242, functions as a signal inhibitor of the TLR-4 pathway actingafter TLR-4 binds with LPS. In septic animal models an improved survivalassociated with decreased levels of inflammatory cytokines has beenshown with the use of this therapy. Furthermore, its use in healthyvolunteers prior to instillation of LPS also resulted in decreasedlevels of inflammatory cytokines when these patients were given an LPSchallenge. In 2010, Rice et al. evaluated TAK-242 in a randomized,placebo-controlled trial of patients with severe sepsis and shock orrespiratory failure. High-dose and low-dose treatment regiments werecompared to placebo with primary endpoints of change in IL-6 level and28-day mortality rate. This trial was terminated after enrollment of 274patients failed to show suppression of IL-6 levels. Evaluation of thetreated patients showed no difference in 28-day mortality compared toplacebo, however, there was a trend toward improved survival in thosewith both shock and respiratory failure who were in the higher treatmentdose cohort. Rice et al., “A randomized, double-blind,placebo-controlled trial of TAK-242 for the treatment of severe sepsis”Critical Care Medicine. 2010; 38(8):1685-1694. It may be that thistherapy could be effective in patients with a higher severity ofillness, as suggested by the trend towards improved survival of thepatients with both respiratory failure and shock. Furthermore, the meantime from onset of shock or respiratory failure to initiation of TAK-242therapy was 19 hours. The dynamic nature of the immune response has beenwell described and it could be postulated that the delay of 19 hours istoo long for the treatment to have the ability to suppress the immuneresponse.

C. Steroid Therapy

Steroids are believed to act early in the inflammatory cascade elicitinga wide range of effects via broad suppression of the immune system andare hypothesized to provide benefit as a supplementary treatment ofsepsis. Steroids function by inhibiting production of compoundsincluding, but not limited to, proinflammatory cytokines (i.e., forexample, TNF-α, IL-1, IL-2, IL-6, and IFN-gamma), chemokines,bradykinins, and eicosanoids. Simultaneously, they may increaseanti-inflammatory mediators (IL-10, IL-1 receptor antagonists, and TNFreceptor antagonists), inhibit inducible nitric oxide synthase, decreasemigration of inflammatory cells to sites of inflammation, and reduce thefunction of inflammatory cells. Rice et al., “Therapeutic interventionand targets for sepsis” Annual Review of Medicine. 2005; 56:225-248. Itis further postulated that steroids increase the expression ofadrenergic receptors in the vasculature. These receptors aredownregulated in septic shock and, theoretically, increasing theirexpression allows the vasculature to respond to the high levels ofcirculating cortisol. Hotchkiss et al., “The pathophysiology andtreatment of sepsis” New England Journal of Medicine. 2003;348(2):138-150.

Initially, studies were done using high-dose steroids (e.g., 30 mg/kgmethylprednisolone) with the goal of broad suppression of the body'soverreactive inflammatory response. Annane et al., “Corticosteroids inthe treatment of severe sepsis and septic shock in adults: a systematicreview” Journal of the American Medical Association. 2009;301(22):2362-2375; Batzofin et al., The use of steroids in the treatmentof severe sepsis and septic shock. Best Practice & Research. 2011;25(5):735-743; Cronin et al., “Corticosteroid treatment for sepsis: acritical appraisal and meta-analysis of the literature” Critical CareMedicine. 1995; 23(8):1430-1439. These studies failed to show benefitand even showed a trend towards harm including increased mortality dueto secondary infections in those treated with steroids, thus, causingsteroid therapy use to decrease in the early 1990s. The use of steroidswas revived in the mid 1990s with the target of treating relativeadrenal insufficiency with the use of replacement, low-doseglucocorticoids. The treatment with low-dose steroids is thought toimprove vascular response to endogenous and exogenous catecholamines viathe upregulation of adrenergic receptors in the vasculature. Whileavoiding the substantial immune system blockade, this lower dose isthought to maintain some anti-inflammatory effects via preventingrelease of proinflammatory cytokines and activation of endothelial cellsand neutrophils to decrease sepsis triggered clotting disorders.

Small studies done in the late 1990s showed trends toward improvement inhypotension and mortality with the low-dose steroid treatment strategy.However, these studies were underpowered to detect clinicallysignificant effects. More recently, two large randomized, controlledtrials have been published that further evaluated the effectiveness ofsteroid therapy. In 2002, a study completed by Annane et al. evaluated300 patients with septic shock and showed improvement of refractoryhypotension and a decrease in absolute mortality in patients withrelative adrenal insufficiency treated with 7 days of hydrocortisone andfludrocortisone. This study also showed that adrenal-sufficient patientsas defined as displaying a response to ACTH gained no benefit andtrended towards harm from glucocorticoids. Annane et al., “Effect oftreatment with low doses of hydrocortisone and fludrocortisone onmortality in patients with septic shock” Journal of the American MedicalAssociation. 2002; 288(7):862-871.

Subsequently, the CORTICUS study published in 2008 by Sprung et al.compared the treatment of 499 septic patients with 11 days ofhydrocortisone versus placebo. Contrary to the Annane study, theCORTICUS trial failed to show an improvement in mortality or reversal ofshock in treated patients, regardless of ACTH response. They did,however, show a faster resolution of shock in those patients who hadshock resolution and were treated with hydrocortisone. Interestingly,they did show an increased incidence of superinfection in those treatedwith steroids. Sprung et al., “Hydrocortisone therapy for patients withseptic shock” New England Journal of Medicine. 2008; 358(2): 111-124.The differences in the outcomes of these trials may be linked to severalkey differences between them. The populations studied included differenttiming of patient enrollment. The Annane study took patients up to 8hours after onset of shock while the CORTICUS study extended theirenrollment up to 72 hours after the onset of shock. Furthermore, theCORTICUS trial included all patients in shock while the Annane studyrestricted their study to only those who were both fluid and vasopressorrefractory. Similarly, the patients in the Annane study weresignificantly more ill at baseline with a higher SAPS score and a highermortality rate in the placebo groups than the CORTICUS trial (65% Annanetrial versus 32% CORTICUS trial). It could be postulated that theirconflicting results are a product of their differing patient populationsas the Annane study evaluated a group of patients with a higher degreeof illness and more refractory shock.

Furthermore, the Annane and CORTICUS trials differed regarding theutility of the ACTH stimulation test. The Annane study showed that ACTHnonresponders were more likely to benefit from steroid therapy while theCORTICUS trial failed to replicate this finding. Given the challenges ofmeasuring cortisol levels and the finding that the Annane trial showedan overall trend toward benefit of steroid therapy, regardless of ACTHresponsiveness, the 2008 Surviving Sepsis Guidelines recommended thatthe ACTH stimulation test not to be used as a tool to guide the use ofsteroid therapy. Known side effects of steroids including hyperglycemia,gastrointestinal bleeding, myopathy, and secondary infection havetempered the enthusiasm for steroid use. The CORTICUS trial, showing noefficacy of steroids reinforced these reservations when it alsodemonstrated an increase in episodes of superinfection with new sepsisand septic shock in those treated with steroids.

Currently, the adult literature has not developed a standard of care inregards to steroid therapy. The Surviving Sepsis Guidelines recommendthe use of steroids only in fluid and vasopressor refractory shock anddo not recommend the use of the ACTH stimulation test based on low-gradeand moderate-grade evidence, respectively. Furthermore, they advisetapering the steroids when the state of shock resolves. Less data existsin regards to the pediatric population and the Surviving SepsisGuidelines base recommendations on a retrospective review. done byMarkovitz et al., “A retrospective cohort study of prognostic factorsassociated with outcome in pediatric severe sepsis: what is the role ofsteroids?” Pediatric Critical Care Medicine. 2005; 6(3):270-274.Consequently, it is possible that corticosteroid use in children withsevere sepsis represents an independent predictor of mortality. However,the nature of the study design does not allow for causal inference andthe Surviving Sepsis Guidelines cite a weak recommendation based onlow-grade evidence for the use of hydrocortisone only in children withcatecholamine resistant shock and suspected or proven adrenalinsufficiency. Ideally, what is needed is a better means of determiningthe population of septic patients which have the best chance to benefitfrom steroid treatment while having the least risk of harm due to theside effects of the therapy.

D. Proinflammatory Cytokine Inhibitor Therapy

Knowledge of the inflammatory cascade and, more specifically,proinflammatory cytokines has allowed specific targets forimmunosuppression including TNF-α and IL-1. TNF-α injection into animalshas been shown to trigger a sepsis-like syndrome including hypotension,activation of the clotting cascade, significant organ dysfunction, andeven death. Furthermore, increasing and persistently elevated levels ofTNF-α are associated with nonsurvival in humans. Qiu et al., “Theevolving experience with therapeutic TNF inhibition in sepsis:considering the potential influence of risk of death” Expert Opinion onInvestigational Drugs. 2011; 20(11): 1555-1564; and Reinhart et al.,“Anti-tumor necrosis factor therapy in sepsis: update on clinical trialsand lessons learned” Critical Care Medicine. 2001; 29(supplement7):S121-S125.

Downstream effects of TNF-α include augmentation of the inflammatorycascade via elevation of multiple cytokine levels and upregulation ofadhesion molecules on leukocytes, platelets, and endothelial cells.TNF-α also stimulates the coagulation system via activation ofthrombotic and fibrinolytic pathways. Despite the deleterious effects ofthis overstimulation, it is evident that TNF-α plays a crucial role inthe immune system because blockage of its activity in animal models hasled to a worsened ability of the animal's immune system to clearmicrobes. Due to its pivotal position in the inflammatory andcoagulation systems that are known to cause the demise in sepsis, TNF-αhas been targeted as a treatment of sepsis in many clinical trials.Although no trial has succeeded in showing an overall improvement usingthis therapy, several studies have identified populations and/orcharacteristics of these patients that may direct future trials.

The first large trial, NORASEPT, was done by Abraham et al. in 1995 andincluded 900 patients with sepsis or septic shock. The NORASEPT trialevaluated an anti-TNF-α monoclonal antibody and failed to show anoverall mortality benefit. However, the subset of patients with septicshock showed a significant improvement in mortality 3 days after druginfusion. In following the patients further, the 28-day mortalitycontinued to show a trend towards improvement but was no longersignificant. Abraham et al., “Efficacy and safety of monoclonal antibodyto human tumor necrosis factor α in patients with sepsis syndrome: arandomized, controlled, double-blind, multicenter clinical trial”Journal of the American Medical Association. 1995; 273(12):934-941. TheINTERSEPT study, published in 1996, focused on evaluation of 420patients with septic shock. This study showed more rapid reversal ofshock and fewer patients with at least one organ failure in survivorswho were treated with the anti-TNF-α monoclonal antibody as comparedwith the placebo group. However, this trial failed to show a differencein mortality. Cohen et al., “INTERSEPT: an international, multicenter,placebo-controlled trial of monoclonal antibody to human tumor necrosisfactor-α in patients with sepsis” Critical Care Medicine. 1996;24(9):1431-1440. This drug was tested in a third trial, NORASEPT II,which also failed to show an improvement in mortality. Abraham et al.,“Double-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shock” The Lancet.1998; 351(9107):929-933.

A trial of an anti-TNF-α antibody fragment, afelimomab, was done byReinhart et al. and published in 1996 that suggested a benefit oftreatment in patients with baseline elevation of IL-6. Reinhart et al.,“Assessment of the safety and efficacy of the monoclonal anti-tumornecrosis factor antibody-fragment, MAK 195 F, in patients with sepsisand septic shock: a multicenter, randomized, placebo-controlled,dose-ranging study” Critical Care Medicine. 1996; 24(5):733-742.Physiologically, this association is plausible as IL-6 levels areconsidered to be a surrogate for overall TNF-α activity due to thelonger half-life of IL-6 compared to the rapidly cleared TNF-α. Thishypothesis was tested in a prospective, randomized placebo-controlledtrial, the RAMSES study of 446 patients with elevated IL-6 levels. Itshowed a nonsignificant trend towards improved survival in those treatedwith afelimomab. Reinhart et al., “Randomized, placebo-controlled trialof the anti-tumor necrosis factor antibody fragment afelimomab inhyperinflammatory response during severe sepsis: the RAMSES study”Critical Care Medicine. 2001; 29(4):765-769. A later study, the MONARCStrial, tested the same antibody fragment in 998 patients with elevatedIL-6 levels and found a trend towards improved survival in treatedpatients as compared to placebo. The risk-adjusted reduction inmortality was 5.8% and corresponded to a relative risk reduction formortality of 11.9%. This study also found a greater reduction in IL-6levels and multiorgan dysfunction score in those treated withafelimomab. The results are also encouraging because patients withhigher IL-6 levels had significantly higher mortality rates in theplacebo group than those with lower IL-6 levels. Thus, this showed thatafelimomab had a greater effect in patients at higher risk of mortality.Panacek et al., “Efficacy and safety of the monoclonal anti-tumornecrosis factor antibody F(ab′)2 fragment afelimomab in patients withsevere sepsis and elevated interleukin-6 levels” Critical Care Medicine.2004; 32(11):2173-2182. In a similar investigation, cytofab, apreparation of polyclonal ovine anti-TNF Fab IgG fragments, was testedin a phase II placebo-controlled randomized clinical trial in 81 septicpatients with shock or two organ dysfunctions. While this study did notshow a difference in mortality, the investigators were able to show anincrease in ventilator-free days, ICU-free days, and a decrease in serumand BAL levels of TNF-α and downstream effects on IL-6 in patientstreated with CytoFab. Rice et al., “Safety and efficacy ofaffinity-purified, anti-tumor necrosis factor-α, ovine fab for injection(CytoFab) in severe sepsis” Critical Care Medicine. 2006;34(9):2271-2281. The persistent trends toward improved survival in theabove studies are encouraging that some patients have the ability tobenefit from immunotherapies. The difficulty lies in determining whichpatients are most likely to benefit.

With a similar mechanism of action, IL-1 is also a target ofimmunotherapies. This proinflammatory cytokine works together with TNF-αto propagate the hyperimmune response of sepsis. Macrophages and othercells naturally produce IL-1 receptor antagonist (IL-ra) in response toIL-1, endotoxin, and various other microbial elements. The IL-1rareversibly binds and competitively inhibits IL-1 receptors. Fisher etal., “Recombinant human interleukin 1 receptor antagonist in thetreatment of patients with sepsis syndrome: results from a randomized,double-blind, placebo-controlled trial” Journal of the American MedicalAssociation. 1994; 271(23):1836-1843. In 1994, Fisher et al. published astudy evaluating the use of IL-1ra in the treatment of 893 patients withsepsis. This study failed to show an overall increase in survival inthose treated as compared to placebo. However, retrospective andsecondary analyses identified a trend of increased survival amongpatients with sepsis as well as an organ dysfunction and/or a predictedrisk of mortality ≧24%. Subsequently, Opal et al. published a trial in1997 focusing on IL-1ra treatment in patients with severe sepsis and/orseptic shock. Disappointingly, this study was halted when just over halfof the proposed enrollment was completed and analysis revealed a lowlikelihood of showing a statistical difference in their primaryendpoint, 28-day mortality. Secondary endpoints showed that thosepatients treated with IL-1ra displayed a nonsignificant trend towardsimprovement of organ dysfunction. The authors postulate that they mayhave had greater success if they were able to identify a more homogenouspopulation. They were also concerned that their treatment was unable tomaintain the necessary 100-10,000 fold excess of IL-1ra relative to IL-1as it is known that stimulation of as few as 5% of the IL-1 receptorstriggers an inflammatory response. Opal et al., “Confirmatoryinterleukin-1 receptor antagonist trial in severe sepsis: a phase III,randomized, double-blind, placebo-controlled, multicenter trial”Critical Care Medicine. 1997; 25(7): 1115-1124. Perhaps furtherevaluation of this drug with the monitoring of levels to ensure completeblockage of the receptors or use of the drug in a more targetedpopulation would provide a better chance for success.

E. Statin Therapy

It is believed that there are many ways which statins have the abilityto affect the immune response in sepsis, however, the exact mechanism oftheir action is unknown. Statins can inhibit the reduction ofhydroxymethyl-glutaryl-CoA to mevalonate which plays a role in synthesisof bile acids, some steroid hormones, and vitamin D. Statins may inhibitvarious other pathways involved in pathophysiology of sepsis includinginhibition of the production of cyclo-oxygenase-2 protein, biosynthesisof ubiquinone which functions in the electron transport chain ofmitochondria, heme-A used in oxygen transport, and prenylation of smallG proteins. It is likely that the alteration of the G-protein pathwayshas the most influential effect as this significantly altersinflammatory cell activation and protein production: Among otherproteins, it is known to inhibit the production of subunits necessaryfor the GTP binding protein Rho. This inhibition has the downstreameffect of production of a decreased amount of inflammatory cytokinessuch as IL-6 and IL-1. Furthermore, hMGCoA-reductase also inducescaspase-dependent apoptosis in smooth muscle cells that may result inless inflammation due to avoidance of necrotic cell death. Bernard G R.,“Statins for acutely hospitalized patients: randomized controlled trialsare long overdue” Critical Care. 2010; 14(2):p. 141.

Data from prospective, randomized-controlled trials evaluating the useof statin therapy in sepsis is lacking. However, multiple observationalstudies show encouraging effects. A large cohort study of more than12,000 critically ill patients was published by Christensen et al. in2010. Results showed that patients on statin therapy immediately priorto ICU admission had a decreased risk of mortality within 30 days and upto 1 year after ICU admission. Given the design of this study, theauthors are unable to infer causation but the results stimulateexcitement for further evaluation of the effects of statin use.Christensen et al., “Preadmission statin use and one-year mortalityamong patients in intensive care—a cohort study” Critical Care. 2010;14(2):p. R29 A large meta-analysis done by Bjorkhem-Bergman et al,published in 2010, evaluated the potential use of statin therapy inbacterial infection. It showed that patients on statin therapy seemed tohave better outcomes including decreased mortality. However, when the 15observational studies were adjusted for publication bias the associationfailed to reach statistical significance. Björkhem-Bergman et al.,“Statin treatment and mortality in bacterial infections—a systematicreview and meta-analysis” PloS one. 2010; 5(5):p. e10702. During thatsame year, Janda et al. focused the evaluation further when theypublished a meta-analysis evaluating statin therapy in severe infectionsand sepsis. This study included 20 trials, mostly cohort studies and onerandomized-controlled trial that demonstrated a protective effectassociated with statin use. The positive outcomes evaluated included30-day mortality, in-hospital mortality, pneumonia-related mortality,bacteremia-related mortality, sepsis-related mortality, and mixedinfection related morality. Again, this study was limited due to theinclusion of mostly cohort studies and significant heterogeneity oftrials. Janda et al., “The effect of statins on mortality from severeinfections and sepsis: a systematic review and meta-analysis” Journal ofCritical Care. 2010; 25(4):656-e7. The one randomized controlled trialin this data set was completed by Tseng et al. and included 80 patientswith aneurysmal subarachnoid hemorrhages. While this study did show animprovement in sepsis-associated mortality, it cautioned that thisfinding was a secondary outcome. Tseng et al., “Effects of acutepravastatin treatment on intensity of rescue therapy, length ofinpatient stay, and 6-month outcome in patients after aneurysmalsubarachnoid hemorrhage” Stroke. 2007; 38(5):1545-1550. Due to thepromising effects of statins, both based on physiologic knowledge and onthe current observational data, phase II and phase III studies arecurrently in progress to evaluate the role of statins in the treatmentof sepsis.

F. Coagulation Cascade Inhibitor Therapy

The extreme activation of the inflammatory system in severe sepsis isaccompanied by a potentially equal stimulation of the coagulationsystem. From an adaptive perspective, this interaction is logical as theactivation of the coagulation system can be envisioned as an effort toisolate the infection with the goal of limiting its spread throughoutthe body.

Various steps of the coagulation pathway have been targeted in thetreatment of sepsis. Tissue factor (TF), a cell surface receptor whoseexpression by endothelial cells and monocytes occurs in the presence ofinflammatory mediators, acts to initiate the extrinsic coagulationpathway. A TF inhibitor was tested in the Phase III trial, OPTIMIST,evaluating its use in 1,754 patients with severe sepsis and this trialfailed to show an improvement in mortality. More concerning, it showed atrend towards harm in those treated concurrently with heparin. Abrahamet al., “Efficacy and safety of tifacogin (recombinant tissue factorpathway inhibitor) in severe sepsis: a randomized controlled trial”Journal of the American Medical Association. 2003; 290(2):238-247.Similarly, antithrombin III (AT III), an anticoagulant, was the subjectof sepsis therapy as well due to the finding of decreased AT II levelsin severe sepsis and the hypothesis that this deficiency contributes tothe hypercoagulation pathophysiology in sepsis. Multiple small studiespublished in the 1990s showed promising results. However, in a phase IIItrial of 2,314 septic patients, they were unable to show a difference inoverall mortality. However, in subgroup analysis, patients not treatedconcomitantly with heparin showed a significant decrease in mortality at90 days while those treated with heparin showed a significantlyincreased risk of bleeding. Warren et al., “Caring for the criticallyill patient. High-dose antithrombin III in severe sepsis: a randomizedcontrolled trial” Journal of the American Medical Association. 2001;286(15):1869-1878. Future investigation of AT III as a treatment forsepsis will need to carefully select their target population to ensureminimal risks for bleeding.

To date, the only drug that has been approved for the treatment ofsevere sepsis is recombinant human activated protein C (rhaPC). It wasinvestigated due to its anti-apoptotic, anti-inflammatory, andanticoagulant effects. It acts via inhibition of factors Va and VIIIawhich results in the prevention of thrombin generation. Downstream, thisdecreases inflammation by reducing mast cell degranulation, plateletactivation, and neutrophil recruitment. Bernard et al., “Efficacy andsafety of recombinant human activated protein C for severe sepsis” NewEngland Journal of Medicine. 2001; 344(10):699-709. The PROWESS trial,published in 2001 spurred great excitement due to its absolute reductionin 28-day mortality by 6.1% in septic patients treated with rhaPC and itwas subsequently approved for use in the most severely ill septicpatients with APACHE scores greater than 25 as this subgroup seemed toderive the most benefit from treatment. Unfortunately, these resultswere not replicated in the PROWESS-SHOCK study and the treatment wasvoluntarily removed from the market by the manufacturer Ranieri et al.,“Drotrecogin alfa (activated) in adults with septic shock” New EnglandJournal of Medicine. 2012; 366(22):2055-2064. The use of rhaPC is notrecommended for use in children based on a study published in 2007 thatevaluated 477 septic children and failed to show an improvement inmortality. Nadel et al., “Drotrecogin alfa (activated) in children withsevere sepsis: a multicentre phase III randomised controlled trial” TheLancet. 2007; 369(9564):836-843

Thrombomodulin (TM), another naturally occurring pathway in thecoagulation system, is currently being targeted in the treatment ofsepsis. TM, produced by endothelial cells, acts upstream in theactivated protein C pathway to sensitize the thrombin receptor leadingto activation of protein C. Yamakawa et al. “Treatment effects ofrecombinant human soluble thrombomodulin in patients with severe sepsis:a historical control study” Critical Care. 2011; 15(3):p. R123. It hasbeen shown that the serum concentration of TM parallels the severity ofcoagulopathy and organ failure in sepsis and decreases as DIC and ARDSimproves. Lin et al., “Serum thrombomodulin level relates to theclinical course of disseminated intravascular coagulation, multiorgandysfunction syndrome, and mortality in patients with sepsis” CriticalCare Medicine. 2008; 36(3):683-689. A control study of 20 patients withsevere sepsis-induced DIC treated with rhTM compared to 45 historicalcontrols showed improved 28-day mortality and improved organ dysfunctionin those treated with rhTM. Ongoing phase 11 studies are in progress toevaluate the efficacy of rhTM.

G. Immunostimulator Therapy

Due to the recognition that sepsis is characterized by a combination ofhyperimmune response and relative immunoparalysis, furtherinvestigations have pursued immunostimulatory strategies. Acontroversial and widely studied therapy is treatment with the use ofpooled serum polyclonal immunoglobulin preparations, IVIG. Although theexact mechanism remains in question, it is thought that theimmunoglobulins coat bacteria, which improves phagocytosis and enhancesneutralization and opsonization causing inactivation of bacterialendotoxins and exotoxins. Furthermore, it is hypothesized that thetreatment alters the release of cytokines and cytokine antagonists byendotoxin and interacts with the complement cascade causing an improvedimmune response in sepsis. Further supporting this strategy is a recentstudy which evaluated 62 adult septic patients and revealed decreasedlevels of immunoglobulins particularly IgG and IgM early in sepsis ascompared to age-matched controls. This was followed by normalization oflevels after 7 days in the majority of patients. Decreased level ofimmunoglobulins was associated with decreased levels of plasma proteinsbut was not associated with a difference in mortality. Venet et al.,“Assessment of plasmatic immunoglobulin G, A and M levels in septicshock patients” International Immunopharmacology. 2011;11(12):2086-2090. In 2007 and 2008, three meta-analyses were publishedthat evaluated the efficacy of polyclonal IVIG in adult patients withsepsis. All three concluded that this therapy improved survival but, dueto small study sizes, heterogeneity, and methodologic limitations of theindividual studies, the three authors recommended large randomized,controlled trials to verify therapeutic efficacy. Kreymann et al., “Useof polyclonal immunoglobulins as adjunctive therapy for sepsis or septicshock” Critical Care Medicine. 2007; 35(12):2677-2685: Laupland et al.,“Polyclonal intravenous immunoglobulin for the treatment of severesepsis and septic shock in critically ill adults: a systematic reviewand meta-analysis” Critical Care Medicine. 2007; 35(12):2686-2692; andTurgeon et al., “Meta-analysis: intravenous immunoglobulin in criticallyill adult patients with sepsis” Annals of Internal Medicine. 2007;146(3):193-203.

A subsequent Cochrane review published in 2010 evaluated 17 trials ofpolyclonal IVIG in adult patients with sepsis. This review was inagreement with the prior meta-analyses and showed a reduction in lessthan 30-day mortality in treated patients. However, the authorsrecommended cautious interpretation of their findings as the majority ofstudies had a small sample size and there was concern for poormethodologic quality. Furthermore, when the trials were restricted tothose with low risk of bias, no reduction in mortality was shown and thestudies that evaluated long-term mortality (greater than 60 days) didnot show an effect. The Cochrane review went on to specify theiragreement with the Kreymann et al. meta-analysis findings that theIgM-enriched formulation of immunoglobulin is also beneficial and eventrended toward a greater effectiveness in the treatment of sepsis. Itwas concluded that polyclonal immunoglobulins appear to be beneficial asadjuvant therapy for sepsis.

The pediatric population stands to reap greater benefit from IVIG due tothe immaturity of B-cells in patients less than 5 years old. In 2005, aprospective case-controlled trial of 100 pediatric patients showed asignificant improvement in length of stay, development of complications,and mortality in septic pediatric patients 1 month-24 months old treatedwith IVIG. El-Nawawy et al., “Intravenous polyclonal immunoglobulinadministration to sepsis syndrome patients: a prospective study in apediatric intensive care unit” Journal of Tropical Pediatrics. 2005;51(5):271-278. Based on the findings of this study, the Surviving SepsisGuidelines recommend consideration of IVIG treatment of pediatricpatients with severe sepsis. However, this recommendation is supportedonly by weak evidence due to low trial quality. IVIG in the neonatalpopulation is equally as controversial as the Cochrane review found noreduction in mortality in septic neonates treated with IVIG while theSurviving Sepsis Guidelines cite that there is evidence to supportimproved mortality in neonates treated with IVIG. A study published byBrocklehurst et al. in 2011, after the publication of the SurvivingSepsis Guidelines and Alejandria's Cochrane review, evaluated over 3,000neonates with sepsis and found no difference in the primary outcomes ofmortality or major disability up to two years of age. Brocklehurst etal., “Treatment of neonatal sepsis with intravenous immune globulin” NewEngland Journal of Medicine. 2011; 365(13):1201-1211.

Other immunostimulatory strategies include cytokine stimulation withgranulocyte-colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), and IFN-gamma. The hypothesizedmechanism of these therapies in nonneutropenic patients is stimulationof bactericidal activity via increased leukocytosis and increasedactivity of granulocytes. Bo et al. published a meta-analysis of 21randomized-controlled trials evaluating G-CSF and GM-CSF in thetreatment of sepsis. This evaluation of a combined 2,380 septic patientsshowed no change in mortality but did show a positive effect of thistherapy on the rate of reversal of infection. They found no differencein adverse events between the groups and recommended further studies toevaluate the efficacy of this therapy. Bo et al., “Granulocyte-colonystimulating factor (G-CSF) and granulocyte-macrophage colony stimulatingfactor (GM-CSF) for sepsis: a meta-analysis” Critical Care. 2011;15(1):p. R58.

G-CSF and GM-CSF may differ in that GM-CSF has additional monocytic andmacrocytic stimulatory affects, inducing monocytic cytokine expressionand antigen presentation via increased expression mHLA-DR theoreticallyresulting in improved adaptive immunity. In the meta-analysis by Bo etal., the data evaluating these two therapies were combined despite theirdiffering mechanisms of action. Furthermore, the studies differedsignificantly on dose as well as on the route of administration andfailed to stratify the patients based on their immunologic state. Giventhe immune-stimulatory mechanism of this therapy, it would be importantto know if the patients being studied are in the hyper or hypoimmunephase of sepsis as this may affect the drug's efficacy. Furthermore, newdata has shown that it is possible to track the efficacy ofimmunostimulatory therapies by measurement of mHLA-DR expression onmonocytes which is decreased in patients with sepsis-associated immunecell dysfunction. Meisel et al. recently showed that patients withsepsis-associated immunosuppression, defined as low monocytic HLA-DRexpression, who were treated with GM-CSF had improvement of monocyticHLA-DR expression when compared to placebo patients. Although this trialincluded only 38 patients, they were able to show shorter duration ofmechanical ventilation as well as shorter ICU and hospital lengths ofstay. Schefold J C., “Immunostimulation using granulocyte- andgranulocyte-macrophage colony stimulating factor in patients with severesepsis and septic shock” Critical Care. 2011; 15(2):p. 136; Meisel etal., “Granulocyte-macrophage colony-stimulating factor to reversesepsis-associated immunosuppression: a double-blind, randomized,placebo-controlled multicenter trial” American Journal of Respiratoryand Critical Care Medicine. 2009; 180(7):640-648.

IFN-gamma has shown similar ability to restore monocytic HLA-DRexpression in septic patients with evidence of monocyte deactivation. Asmall study done by Docke et al. showed that IFN-gamma treatment inseptic patients with low monocytic HLA-DR expression resulted inrestoration of monocyte function as measured by improved TNF-α secretionresulting in clearance of sepsis in 8 of 9 patients. Docke et al.,“Monocyte deactivation in septic patients: restoration by IFN-gammatreatment” Nature Medicine. 1997; 3(6):678-681.

The results of these studies are encouraging that immunostimulation maybe an effective way to treat the subset of septic patients who are inthe immunoparalysis phase of their disease.

V. Sepsis Proteomic Expression Profiles: Age-Related Treatment

In one embodiment, the present invention contemplates a method fordeveloping efficient therapeutic strategies for patients with sepsis toslow inflammatory response and reduce mortality rates. As detailedabove, antimicrobial treatments and early goal-directed therapy are usedin the management of sepsis. Carvalho et al., Journal of Pediatrics79:S195 (2003); and Hotchkiss et al., New England Journal of Medicine348:138 (2003). However, these strategies in clinical practice arelimited. For example, the choice of a specific antimicrobial treatmentcan be influenced by the infection site and molecules released frommicroorganisms may exacerbate inflammatory response. Other therapies,including but not limited to, glucocorticoids, recombinanthuman-activated protein C, steroids, intravenous Ig, continuous renalreplacement therapy, proinflammatory cytokine inhibitors, antioxidantsupplementation have failed to provide an effective treatment forsepsis. Overall, more than forty (40) clinical trials of therapiestargeted at septic patients have failed to lead to a currentFDA-approved drug for the universal treatment of sepsis. Annane et al.,British Medical Journal 329:480 (2004); Casserly et al., Critical CareMedicine 40:1417 (2012); Patel et al., American Journal of Respiratoryand Critical Care Medicine 185:133 (2012); Turgeon et al., Annals ofInternal Medicine 146:193 (2007); Joannidis, M., Seminars in Dialysis22:160 (2009); Berger et al., Critical Care Medicine 35:S584 (2007):Ward et al., Critical Care Research and Practice 2012:8 (2012);Hotchkiss et al., The Lancet Infectious Diseases 13:260 (2013); Wu etal., Shock 21:210 (2004).

The lack of current universal treatment for sepsis can be understood dueto the many challenges associated with this condition. Diagnosis ofsepsis is complicated by its differential presentation between patients(i.e., degree and nature of clinical symptoms can vary tremendously). Inaddition to patient heterogeneity, there is also temporal heterogeneitywithin individual patients. An et al., Critical Reviews in BiomedicalEngineering 40:341 (2012). The timing of the diagnosis plays a roleregarding the initiation of therapy, however, this is difficult becausecell culture determination assays can take as long as 48 h and somepatients have symptoms for a day prior to hospital admission. Rivers etal., Shock 39:127 (2013). Furthermore, in more than half of sepsiscases, clinical symptoms may persist without a positive culture.Carrigan et al., Clinical Chemistry 50:1301 (2004).

Monitoring of specific biomarkers (e.g., tumor necrosis factor (TNF)-αinterleukin (IL)-1, IL-6, IL-8, and IL-10, procalcitonin, C-reactiveprotein, and others) have yielded conflicting results with regards tosensitivity, specificity, and effectiveness in both adults and neonates.Rivers et al., Shock 39:127 (2013). Furthermore, in more than half ofsepsis cases, clinical symptoms may persist without a positive culture.Carrigan et al., Clinical Chemistry 50:1301 (2004). Because many ofthese molecules also play a role in immune response and inflammation, asingle readout of any specific biomarker can be misleading. Biomarkerreadouts are heavily time-dependent as at least one acuteproinflammatory response occurs within the first 24 h after infection.Rivers et al., Shock 39:127 (2013). Although it is not necessary tounderstand the mechanism of an invention, it is believed that thesechallenges to developing new therapeutics and treatment strategiesregarding sepsis diagnosis, prognosis, and real-time response totreatment of sepsis can be best facilitated by understanding themolecular mechanisms of this condition.

Although it is not necessary to understand the mechanism of aninvention, it is believed that the sepsis proteomic expression profileddisclosed herein imply that age is an important factor affecting apatient response to sepsis. For example, hyper- and hypo-inflammatoryresponse is found in the younger and older severe sepsis patients,respectively. These findings suggest that doctors need to be cautiouswhen interpreting clinical data from sepsis patients at different ages.For example, high plasma concentrations of CRP may lead to thedevelopment of severe sepsis in younger CAP patients but low CRPconcentrations may indicate poor outcome in older CAP patients. Theseobservations also suggest that different treatments need to be performedbased on the ages of sepsis patients.

This differential strategy to treat sepsis may be related to aphenomenon termed immunosensence. Immunosenescence usually refers to agradual deterioration of the immune system during aging. It is widelyaccepted that immunosenescence is characterized by diminished immuneresponse, low-grade inflammation, and increased propensity forautoimmunity. These changes in the immune system increase the risk andmortality of diseases among the elderly. Poor response toimmunotherapies as a result of immunosenescence can further worsen thissituation. In particular, vaccination is an important intervention fordisease control and has been used effectively in children and youngeradults. Goronzy et al., Nature Immunology 14:428 (2013). A fundamentalunderstanding of immunosenescence would be helpful for the preventionand treatment of aging-related diseases. For example, genomics,transcriptomics, proteomics, and metabolomics can give insight tomolecular mechanisms of immunosenescence and its contribution todeveloping proper and effective treatments for age-related diseases(e.g., for example, severe sepsis).

The data presented herein show that there are specific protein pathwaysthat are differentially expressed between younger and older patientswith sepsis. Consequently, this may explain the above described failureof those in the art to identify conclusive sepsis biomarkers. Forexample, tests of sepsis or to predict sepsis severity have beenlimited. In one report, an AUC of about 0.73 for prediction of mortalityusing a 6-marker panel. Wong et al., “A Multibiomarker-Based OutcomeRisk Stratification Model for Adult Septic Shock” Critical Care Medicine42(4):781-789 (2014). Procalcitonin is approved for risk assessment insepsis but its performance isn't much better. Although it is notnecessary to understand the mechanism of an invention, it is believedthat sepsis may have different biomarker patterns between older ascompared to younger adults leading to different biomarker patterns.

In one embodiment, the present invention contemplates that thedifferentially expressed protein pathways between older and youngersepsis patients represent age-specific drug targets. Although it is notnecessary to understand the mechanism of an invention, it is believedthat giving young and old the same therapies could harm one or theother.

In one embodiment, the present invention contemplates specific drugtargets for the treatment of an older sepsis patient. In one embodiment,the drug target comprises a hepatic stellate cell activation pathway. Inone embodiment the drug target comprises an actin cytoskeleton signalingpathway. In one embodiment, the method further comprises a combinationtreatment of the specific drug targent with a conventional sepsistreatment. In one embodiment, the conventional treatment includes, butis not limited to, antibiotics, inflammatory mediator inhibitors,steroids, proinflammatory cytokine inhibitors, statins, coagulationcascade inhibitors and/or immunostimulators.

In one embodiment, the present invention contemplates specific drugtargets for the treatment of a younger sepsis patient. In oneembodiment, the drug target comprises a blood factor coagulationpathway. In one embodiment, the drug target comprises an extrinsicprothrombin activation pathway. In one embodiment, the drug targetcomprises an intrinsic prothrombin activation pathway. In oneembodiment, the method further comprises a combination treatment of thespecific drug targent with a conventional sepsis treatment. In oneembodiment, the conventional treatment includes, but is not limited to,antibiotics, inflammatory mediator inhibitors, steroids, proinflammatorycytokine inhibitors, statins, coagulation cascade inhibitors and/orimmunostimulators.

VI. Pharmaceutical Compositions/Formulations

The present invention further provides pharmaceutical compositions(e.g., comprising the compounds described above). The pharmaceuticalcompositions of the present invention may be administered in a number ofways depending upon whether local or systemic treatment is desired andupon the area to be treated. Administration may be topical (includingophthalmic and to mucous membranes including vaginal and rectaldelivery), pulmonary (e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratacheal, intranasal, epidermaland transdermal), oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. Agents that enhance uptake of oligonucleotides atthe cellular level may also be added to the pharmaceutical and othercompositions of the present invention. For example, cationic lipids,such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerolderivatives, and polycationic molecules, such as polylysine (WO97/30731), also enhance the cellular uptake of oligonucleotides.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models or based on the examples described herein. Ingeneral, dosage is from 0.01 μg to 100 g per kg of body weight, and maybe given once or more daily, weekly, monthly or yearly. The treatingphysician can estimate repetition rates for dosing based on measuredresidence times and concentrations of the drug in bodily fluids ortissues. Following successful treatment, it may be desirable to have thesubject undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the compound is administered in maintenancedoses, ranging from 0.01 μg to 100 g per kg of body weight, once or moredaily, to once every 20 years.

EXPERIMENTAL Example I Expression Profile Patent Population

The data presented herein was collected during a nested case-controlstudy using patients enrolled in the GenIMS study. Kellum et al.,“Understanding the Inflammatory Cytokine Response in Pneumonia andSepsis: Results of the Genetic and Inflammatory Markers of Sepsis(GenIMS) Study” Arch Intern Med 167:1655-1663 ((2007).

A total of 39 patients comprised the four experimental groups:

1) patients 50-65 years old who did not develop severe sepsis (YC);

2) patients 70-85 years old who did not develop severe sepsis (OC);

3) patients 50-65 years old who developed severe sepsis (YS), and

4) patients 70-85 years old who developed severe sepsis (OS).

To ensure that differences in immune response are attributed to age andnot to ethnic differences or underlying chronic diseases, only whitesand matched patients according to chronic disease burden were included.Approval for the participation of human patients was obtained by theInstitutional Review Board of the University of Pittsburgh and otherparticipating sites.

Example II Blood Sample Collection and Processing

Plasma samples were obtained from patients at initial presentation andprior to most interventions to ensure that differences in immuneresponse are not affected by therapeutic strategies. Crude human plasmawas prepared and analyzed as described in FIG. 1.

Multiple Affinity Removal System (MARS) columns were used to depletehigh-abundance proteins. Unbound proteins on the MARS column weresubject to trypsin digestion and modified with an iTRAQ reagent. The Hu6 MARS column (Agilent; Santa Clara, Calif.) depletes serum albumin,IgG, α1-antitrypsin, IgA, transferrin, and haptoglobin proteins. Aninjection amount of 60 μL of crude plasma was applied to the MARS columnand after the initial depletion flow-through fractions were concentratedwith a 5K molecular weight cutoff concentrator (Agilent) at 4695 g for1.5 hours.

Modified peptides were combined, fractionated with Strong CationExchange (SCX), and thirteen fractions were collected and analyzed byLC-MS/MS on a linear ion trap-Orbitrap Velos MS. Collision induceddissociation (CID) and high energy C-trap dissociation (HCD) were usedfor gas-phase fragmentation of peptides. Proteome Discoverer 1.2 wasused to process data files, perform SEQUEST searches, and extract iTRAQinformation. Plasma from CAP patients was used to optimize the workflow.

Samples were then stored at −80° C. or re-injected onto the MARS columnfor tandem MARS depletion. The second flow-through fractions (hereafterreferred to as TMD) were concentrated and protein concentrations weremeasured using the BCA protein assay.

TABLE 8 Protein amounts recovered from crude plasma in the unboundfractions from MD¹ and TMD² Sample Protein mass, μg % depletion EF^(#),-fold* Crude 4416 / / MD 527 ± 52 88.1  8.4 TMD 343 ± 54 92.2 12.8^(#)enrichment factor, *relative to crude plasma, ¹MARS-Depletion,²Tandem MARS-Depletion

Example III Protein Digestion

Protein was denatured with an extraction buffer (0.2 M Tris, 8 M urea,10 mM CaCl2, pH 8.0), reduced with 1:40 molar excess of dithiothreitol(DTT) for 2 h at 37 □C, and then alkylated with 1:80 molar excess ofiodoacetamide (IAM) for 2 h on ice. The alkylation reaction was quenchedby adding 1:40 molar excess of Cysteine and the mixture was incubated atroom temperature for 30 min. Tris buffer (0.2 M Tris, 10 mM CaCl2, pH8.0) was added to dilute the urea concentration to 2 M. Each sample wasincubated with bovine TPCK-heated trypsin at 1:50 substrate:enzyme ratiofor 24 h at 37° C.

Example IV iTRAQ Labeling

Digested samples were desalted with an HLB cartridge (Waters; Milford,Mass.) and dried by centrifugal evaporation. Each sample was labeledwith an iTRAQ reagent following the manufacturer's protocol (AppliedBiosystems; Foster City, Calif.) with slight modifications. Briefly,each iTRAQ reagent was solubilized with 70 μL ethanol and transferred topeptide mixtures. After 1.5 h of incubation, the reaction was quenchedwith water. Labeled samples were mixed in 1:1:1:1 ratios for iTRAQreagents that generate reporter ions at m/z 114:115:116:117,respectively.

Example V SCX Fractionation

SCX fractionation was carried out on a PolySulfoethyl A 100 mm×2.1 mm, 5μm, 200 Å column (The Nest Group, Inc.; Southborough, Mass.) withbuffers as follows: mobile phase A was 5 mM monopotassium phosphate (25%v/v acetonitrile, pH 3.0), and mobile phase B was 5 mM phosphate, 350 mMpotassium chloride, (25% v/v acetonitrile, pH 3.0). Dried iTRAQ labeledsamples were resuspended in 300 μL of mobile phase A and injected ontothe SCX column. The gradient for SCX was: 0-3 min, 0% B; 3-45 min, 0-75%B; 45-50 min, 75-100% B; 50-55 min, 100% mobile phase B; 55-56 min,100-0% B; 56-106 min, 0% B. Thirteen SCX fractions were collected andeach fraction was desalted with an HLB cartridge (Waters).

Example V I LC-MS/MS Analysis

Online desalting and reversed phase chromatography was performed with aNano-2D-LC system equipped with an autosampler (Eksigent; Dublin,Calif.). Mobile phase A and B for these analyses were 3% (v/v)acetonitrile with 0.1% formic acid and 100% (v/v) acetonitrile with 0.1%formic acid, respectively. SCX fractions (5 μL) were loaded onto atrapping column (100 μm i.d.×2 cm), which was packed in-house with C18200 Å 3 μm stationary phase material (Michrom Bioresource Inc.; Auburn,Calif.) at 3 μL-min⁻¹ in 3% mobile phase B for 3 min. After desalting,the sample was loaded onto an analytical column (75 Mm i.d.×13.2 cm),which was packed in-house with C18 100 Å 3 μm stationary phase material(Michrom Bioresource Inc.). The gradient was as follows: 0-5 min, 10%mobile phase B; 5-75 min, 10-30% B; 75-95 min, 30-60% B; 95-100 min,60-90% B; 100-105 min, 90-10% B; 110-120 min, 10% B. The LC eluent wasanalyzed with positive ion nanoflow electrospray using a LTQ-OrbitrapVelos mass spectrometer (Thermo-Fisher Scientific, Waltham, Mass.).Data-dependent acquisition parameters were as follows: the MS surveyscan in the Orbitrap was 60,000 resolution over 300-1800 m/z; the topsix most intense peaks in the MS survey scan were isolated andfragmented with CID and HCD; CID was performed in the ion trap withnormalized collision energy 35%; HCD was recorded in the Orbitrap withnormalized collision energy 45% and 7,500 resolution; dynamic exclusionwas enabled for 61 seconds and a repeat count of 2 for a duration of 60seconds was allowed and selected ions were placed on an exclusion listfor 61 seconds. Each SCX fraction was subject to triplicate LC-MS/MSanalysis.

Example VII Data Analysis

“.RAW” files were analyzed with Proteome Discoverer 1.2 software(Thermo). Both CID and HCD spectra were used to obtain sequenceinformation against the Uniprot human database (Apr. 25, 2010, 20,295sequences). Sequest search parameters were as follows: two maximumtrypsin miscleavages; precursor mass tolerance 10 ppm; fragment masstolerance 0.8 Da; static modifications were iTRAQ-4plex/+144.102 Da(N-terminus, Lys), and carbamidomethyl modification/+57.021 Da (Cys);dynamic modification of iTRAQ-4plex/+144.102 Da (Tyr). Decoy databasesearching was employed to generate medium (p<0.05) and high (p<0.01)confidence peptide lists. All the peptides with medium and highconfidence were used to identify and quantify proteins. Only proteinswith at least two spectral counts in a technical replicate wereconsidered for further analysis.

Example VIII Statistics

Coefficient of variation (CV) values were calculated for reporter ionratios (e.g., 115/114, 116/114, and 117/114) of proteins quantified inat least six iTRAQ experiments. The mean CV value across the iTRAQexperiments was calculated and used as the total biological variation,Sb. The technical variation, St, was calculated for proteins quantifiedin at least two LC-MS/MS analyses within an individual iTRAQ experiment.The relation between the fold change (F), random variation (S),biological variation (Sb), and technical variation St is expressed bythe formula:

$\begin{matrix}{n = {2\frac{\left( {2 + T} \right)^{2}S^{2}}{\left( {F - 1} \right)^{2}}}} & (1) \\{S^{2} = {2\left( {\frac{S_{b}^{2}}{n} + \frac{S_{t}^{2}}{nm}} \right)}} & (2)\end{matrix}$

Horgan, G. W., “Sample size and replication in 2D gel electrophoresisstudies” Journal of Proteome Research 6:2884-2887 ((2007). Thequantities Z and T depend on the power of the test (i.e., 0.8) and thesignificance level (i.e., 0.05), respectively. n and m, the number ofbiological and technical replicates, respectively, were set to 10 and 3for these studies, such that formula (1) and (2) approximates to:

$\begin{matrix}{10 = {20\frac{S^{2}}{\left( {F - 1} \right)^{2}}}} & (3) \\{S^{2} = {\frac{S_{b}^{2}}{10} + \frac{S_{t}^{2}}{30}}} & (4)\end{matrix}$

Solve formulas (3) and (4),

$\begin{matrix}{F = {\left( \frac{{3\; S_{b}^{2}} + S_{t}^{2}}{15} \right)^{1/2} + 1}} & (5)\end{matrix}$

Based on this power analysis, the calculated fold-change cutoff that wasapplied to these data is ˜1.30. Filter criteria were applied to generatea list of statistically significant differentially-expressed proteins asfollows: 1) proteins identified and quantified in at least sixexperiments, 2) CV values ≦0.60, and 3) fold-change cutoff ≧1.30 or≦0.77.

Example IX Western Blotting Analyses

The changes in the expression of C-reactive protein (CRP),apolipoprotein CIII (Apo CIII), and fibrinogen alpha chain (FAC) weresubject to Western blot analysis. Twenty μg of protein was denatured inan appropriate sample buffer and electrophoretically separated on aCriterion precast gel (Biorad Laboratories; Hercules, Calif.) at 140 V.Proteins from the gel were transferred onto a nitrocellulose membranepaper using a Fast-Transfer Blot System (Biorad). Blots were washedthree times in Wash blot. BSA blocking solution (3%) was added to themembrane and incubated on a rocker for 2 h. A 1:5000 dilution of mousemonoclonal anti-CRP primary antibody (Sigma Aldrich; St. Louis, Mo.),1:5000 dilution of rabbit polyclonal anti-Apo CUIII primary antibody(Abcam; Cambridge, Mass.), or 1:2500 dilution of rabbit monoclonalanti-FAC primary antibody (Abcam) was added and incubated at 4° C.overnight. The blot was rinsed and incubated with a 1:7500 dilution ofanti-mouse or anti-rabbit IgG alkaline phosphatase secondary antibody(Sigma Aldrich) for 1 h on a rocker. The blot was rinsed andcolorometrically developed using 0.51 mM5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP) and 0.24 mMnitrotetrazolium blue (NBT). The dried blot was scanned using a Canonscanner, saved as a “.TIFF” file, and densitometry analyses carried outwith Scion Image Software. Within each experiment, the intensity for thesample from each group was normalized to the total blot intensity andused to generate mean and standard deviation values.

Example X Ingenuity Pathway Analysis

Differentially-expressed proteins were analyzed using Ingenuity PathwayAnalysis (IPA, www.ingenuity.com) to generate a list of pathways thatare statistically relevant (p<0.05).

1. A method, comprising: a) providing; i) at least one biological sample derived from an elderly patient suspected of having an infection; ii) at least one peptide isolated from said at least one biological sample, wherein said at least one isolated peptide comprises at least one isobaric tag for relative and absolute quantitation (iTRAQ) reporter ion; iii) a system comprising a liquid chromatography column and a mass spectrometer; and iv) a control proteomic pathway expression profile; b) contacting said at least one iTRAQ-peptide with said system to create at least one iTRAQ-peptide analysis spectrum; c) processing said at least one iTRAQ-peptide analysis spectrum to create a sepsis proteomic pathway expression profile, wherein said sepsis proteomic pathway expression profile comprises at least one over-expressed protein pathway as compared to said control proteomic pathway expression profile; and d) diagnosing said patient with severe sepsis upon identification of said over-expressed protein pathway.
 2. The method of claim 1, wherein said over-expressed protein pathway is a liver retinoid X receptor activation pathway.
 3. The method of claim 2, wherein said liver retinoid X receptor activation pathway is about ten-fold over-expressed as compared to said control proteomic pathway expression profile.
 4. The method of claim 1, wherein said over-expressed protein pathway is an acute phase response signaling pathway.
 5. The method of claim 4, wherein said acute phase response signaling pathway is about eight-fold over-expressed as compared to said control proteomic pathway expression profile.
 6. The method of claim 1, wherein said over-expressed protein pathway is an atherosclerosis signaling pathway.
 7. The method of claim 6, wherein said atherosclerosis signaling pathway is about seven-fold over-expressed as compared to said control proteomic pathway expression profile.
 8. The method of claim 1, wherein said over-expressed protein pathway is an interleukin-2 signaling pathway.
 9. The method of claim 8, wherein said interleukin-2 signaling pathway is about seven-fold over-expressed as compared to said control proteomic pathway expression profile.
 10. The method of claim 1, wherein said over-expressed protein pathway is a nitric oxide/oxygen reactive species pathway.
 11. The method of claim 10, wherein said nitric oxide/oxygen reactive species pathway is about seven-fold over-expressed as compared to said control proteomic pathway expression profile.
 12. The method of claim 1, wherein said over-expressed protein pathway is a clathrin-mediated endocytosis signaling pathway.
 13. The method of claim 12, wherein said clathrin-mediated endocytosis signaling pathway is about seven-fold over-expressed as compared to said control proteomic pathway expression profile.
 14. The method of claim 1, wherein said over-expressed protein pathway is an lipopolysaccharide/interleukin-1 retinoid X receptor inhibition pathway.
 15. The method of claim 14, wherein said lipopolysacharride-interleukin-1 retinoid X receptor inhibition pathway is about three-fold over-expressed as compared to said control proteomic pathway expression profile.
 16. The method of claim 1, wherein said over-expressed protein pathway is a farnesoid X receptor activation pathway.
 17. The method of claim 16, wherein said farnesoid X receptor activation pathway is about two-fold over-expressed as compared to said control proteomic pathway expression profile.
 18. The method of claim 1, wherein said over-expressed protein pathway is a hepatic stellate cell activation pathway.
 19. The method of claim 18, wherein said hepatic stellate cell activation pathway is about two-fold over-expressed as compared to said control proteomic pathway expression profile.
 20. The method of claim 1, wherein said over-expressed protein pathway is an actin cytoskeleton signaling pathway.
 21. The method of claim 20, wherein said actin cytoskeleton signaling pathway is about two-fold over-expressed as compared to said control proteomic pathway expression profile. 22-87. (canceled) 